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Genetic toxicity in vitro

Description of key information

No genetic toxicity study with zinc m-toluate is available, thus the genetic toxicity will be addressed with existing data on the individual moieties zinc and m-toluate.

Zinc m-toluate is not expected to be genotoxic, since the two moieties zinc and m-toluate have not shown gene mutation potential in bacteria and mammalian cells as well as in in vivo clastogenicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No genetic toxicity study with zinc m-toluate is available, thus the genetic toxicity will be addressed with existing data on the individual moieties zinc and m-toluate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Zinc

Several in vitro studies and two in vivo studies are available on the genotoxicity of zinc oxide. Data on other zinc compounds have also to be taken into account, as the basic assumption is made that after intake all zinc compounds (including metallic zinc) are changed (at least in part) to the ionic species and that it is this zinc cation that is the determining factor for the biological activities of the zinc compounds.

The genotoxicity of soluble and slightly soluble zinc compounds have been extensively investigated in a wide range of in vitro and in vivo studies. The in vitro investigations included non-mammalian and mammalian test systems covering the endpoints of gene mutation, chromosomal aberrations, sister chromatide exchange, unscheduled DNA synthesis (UDS), as well as cell transformation. Available in vivo genotoxicity assays included the micronucleus test and chromosomal aberration test.

The investigated zinc compounds did not increase the mutation frequencies in bacterial systems. Zinc oxide was consistently negative in the Ames test. There was some evidence that zinc oxide induced in the absence of metabolic activation the formation of mutation colonies. Several reviewers noted, however, that these mutations were observed at cytotoxic concentrations and that the analysis did not distinguish between big and small colonies which could be caused by gene mutation or chromosomal aberrations (Thompson et al.,1989, WHO, 2001; EU RAR, 2004; MAK, 2009).

Conflicting information was further found when zinc compounds were examined for their potential to induce chromosomal aberrations or sister chromatide exchange in mammalian cell systems or when evaluated in the cell transformation assay. Positive as well as negative results were obtained in these cell systems with either soluble or slightly soluble zinc compounds. In those studies where, chromosomal aberrations or sister chromatide exchange has been observed, these were generally considered to be weak and occurred only at high, often cytotoxic concentrations. Moreover, these positive in vitro findings have also to be seen in context of the impact that changes in zinc levels can have on cell system processes that are controlled by a strict metal homeostasis. A change of this metal homeostasis due to increased zinc levels, may lead to a binding of zinc to amino acids like cystein and therefore to an inhibition of certain enzymes. This can lead to interactions with the energy metabolism, signal transmission and apoptotic processes which can lead to the observed clastogenic or aneugenic effects in in vitro systems (EU RAR, 2004; MAK, 2009).

In addition to above mentioned in vitro investigations, zinc compounds have also been studied in in vivo studies including the micronucleus test and chromosomal aberration test. The zinc compounds were negative in both assays.

 

The German MAK committee reviewed the existing in vivo evidence and concluded that particularly those studies indicating clastogenic effects involved a lot of methodological uncertainties which do not allow overruling those in vivo studies which did not provide any evidence for chromosomal aberrations in vivo. Moreover, the Dutch rapporteur of EU risk assessment of zinc compounds under the EU existing substance legislation considered the positive in vitro findings for chromosomal aberration and SCE assays to be overruled by the overall weight of evidence of negative in vivo tests for this endpoint (EU RAR, 2004).

 

The overall weight of the evidence from the existing in vitro and in vivo genotoxicity assays suggests that zinc compounds do not have biologically relevant genotoxic activity. This conclusion is in line with those achieved by other regulatory reviews of the genotoxicity of zinc compounds (WHO, 2001; SCF, 2003; EU RAR, 2004, MAK, 2009). Hence, no classification and labelling for mutagenicity are required.

 

m-toluate

m-toluic has been tested in bacterial reverse mutation assay, in a gene mutation test, and in a cytogenicity test. All tests showed a negative response, thus, m-toluic does not require classification for mutagenic properties.

 

Genetic toxicity – in vitro results 

Gene mutation in bacterial test systems

In the NTP study (NTP, 1992) m-toluic acid was assessed in regard to its mutagenic potential using the Ames test. Five Salmonella typhimurium strains (TA97, TA98, TA100, TA1535 and TA1537) were tested at 5 different concentrations (33-3333 µg/plate) using the preincubation method. The cells were either tested without metabolic activation or in presence of either induced male Sprague-Dawley rat liver S9 (10 % and 30 %) or induced Syrian hamster liver S9 (10 % and 30 %). the test substance was not tested at the recommended top dose (OECD 471; EU: B.13/B.14), although no signs of precipitation and cytotoxicity were observed. In two independent experiments, the treated tester strains showed no increased revertant colony number under the conditions tested.

This negative result was confirmed and further extended in a GLP study (Mitsubishi Chemical Safety Institute (Japan); 1999) using two different bacterial genera. A preincubation test was performed using four Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and one Escherichia coli strain (WP2uvrA). The cells were treated either without metabolic activation at six concentrations (156-5000 µg/plate) or with metabolic activation at six concentrations, ranging from 313-5000 µg/plate. m-toluic acid was tested up to the recommended top dose, however, precipitation and cytotoxicity (decrease of revertant number of over 50%) were observed in combination with metabolic activation at the highest dose applied. Additionally, cytotoxicity occurred in Salmonella tester strains TA100, TA1535, and TA1537 at a dose of 2500 µg/plate in presence of a metabolic activation system. Though precipitation and cytotoxicity were observed, the study is compliant with the current guidelines (OECD 471; EU: B.13/B.14) and is well-suited for risk assessment purposes. None of the triplicates showed an increase in the revertant colony number, when compared to controls both in presence and absence of a metabolic activation system.

 

Gene mutation in mammalian cells

In a GLP study (Hargreaves, 2018), the mutagenic potential of m-toluic acid was evaluated at the hprt locus in mouse L5178Y lymphoma cells. The cells were tested at eight concentrations (50-1362 µg/mL), up to highest concentration recommended (equivalent to 10 mM; OECD TG476 and EU: B.17), either in absence or in presence of a metabolic activation system (S-9 rat liver). The cells were incubated for 3 hours and mutant frequency was scored after 12 days in duplicate experiments. Neither significant increases nor positive linear trends in mutant frequency were observed, independent of metabolic activation status. Thus, the test substance did not induce mutations at the hprt locus in mouse lymphoma cells being exposed up to top concentrations recommended by current regulatory testing guidelines.

 

Chromosomal aberrations (CA) in mammalian cells

Only one in vitro cytogenicity study is present, which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA ‘Guidance on Information Requirements and Chemical Safety Assessment – Chapter R.7a’:

Mitsubishi Chemical Safety Institute (Japan), 1999: CHL/IU (Chinese hamster lung) cells were treated in a ‘short time exposure’ experiment at concentration of 250, 500, 1000 and 2000 µg/mL with and without S9, and additionally, in a second ‘continuous exposure’ experiment either for 24 hours at 250, 500, 1000 and 2000 µg/mL or for 48 hours at 62.5, 125, 250, 500 and 1000 µg/mL. Eventually, a confirmation test was performed at either 1000, 1500 and 2000 µg/mL without S9 or 500, 750 and 1000 µg/mL with S9. Positive results were found, in absence of S9, in the ‘continuous exposure’ experiment (exposure for 48 hours) and in the confirmation test at concentrations of 1000 and 2000 µg/ml, respectively. The authors concluded that the test substance induces elevated CA frequencies. However, following methodological deficiencies hamper the interpretation of these findings:

-Tests have been conducted partially above limit dose of 10 mM (equivalent to 1361 µg/mL for m-toluic acid), namely 1500 and 2000 µg/mL;

- Eventually, less than 3 test concentrations were analysable due to cytotoxicity;

- Furthermore, other confounding factors are noticed: The pH of cell culture medium was affected by test substance, the lowest pH observed while inducing chromosomal aberrations was at pH 6.2; the test substance precipitated at test concentrations of 1000 µg/mL and above; the culture medium changed to yellow colour at test concentrations of 1000 µg/mL and above.

 

Genetic toxicity – in vivo somatic cell results

Micronucleus (MN) test

A GLP study (Watabe, 2002 (Mitsubishi Chemical Safety Institute (Japan)) has been conducted to measure MN induction in bone marrow erythrocytes upon m-toluic acid treatment. Five male SD (Cry: CD (SD), IGS) rats per group received, within 24 hours, two oral gavages to reach total doses of 500, 1000, and 2000 mg/kg m-toluic acid. The top dose was chosen based on the findings of a prior single dose toxicity study showing a LD50of greater than 2000 mg/kg. 24 hours after treatment, 2000 polychromatic erythrocytes were scored for MN occurrence. No significant increase in MN induction was observed, except for one rat showing a highly significant increased MN number at a dose of 500 mg/kg. However, this finding was not reproduced in a second confirmation test, in which five animals were exposed to 125, 250, and 500 mg/kg m-toluic acid. The confirmation test showed no elevated MN levels at all doses tested, and consequently, the prior positive finding was proven as chance finding. Concurrent positive and negative controls constituted the study being valid. Therefore, m-toluic acid is concluded to induce no micronucleation under the conditions tested.

 

Discussion

The mutagenic potential of m-toluic acid was evaluated in three independent studies, including two studies which are GLP compliant. The test substance was tested in two in vitro studies (NTP, 1992; and MHLW, 1999) exploiting bacterial test systems and in one GLP study (Hargreaves 2018) using mammalian cells. All of the studies showed that m-toluic acid does not have potential to induce mutations under the conditions tested. Thus, it is assessed to be non-mutagenic in in vitro systems.

Two cytogenicity studies were performed to evaluate the potential of m-toluic acid to induce chromosomal aberrations, both of them were GLP compliant. In the first study (Mitsubishi Chemical Safety Institute (Japan), 1999), an in vitro CA test was performed. However, due to significant methodological deficiencies this study is not suitable for risk assessment purposes. Furthermore, a second cytogenicity study (Watabe, 2002) showed no m-toluic acid-induced clastogenicity, when exploiting an in vivo MN test in rats. Consequently, findings of a possible clastogenic potential indicated by the in vitro CA study could not be confirmed.  

According to Regulation (EC) No 1907/2006 Annex VIII 8.4.2. column 1, an in vitro cytogenicity study is a standard information requirement. However, according to the specific rules for adaption from column 1, in column 2 is clearly stated that the in vitro cytogenicity test could be replaced by an in vivo cytogenicity test provided that the data is adequate. The in vivo MN study (Watabe, 2002) present does satisfactorily meet the requirements according to Regulation (EC) No 440/2008 EU: B.12 and OECD TG474. Thus, according to Regulation (EC) No 1907/2006 Annex VIII 8.4.2., this in vivo cytogenicity study could be used as substitute for the in vitro cytogenicity test required. Moreover, according to the ECHA ‘Guidance on Information Requirements and Chemical Safety Assessment – Chapter R.7a’, in vivo studies indicate a higher degree of reliability, and thus, they are more appropriate as surrogate for human health risk assessment. Under these conditions m-toluic acid is considered to induce no cytogenic damage.

Zinc m-toluate

Zinc m-toluateis not expected to be genotoxic, since the two moieties zinc and m-toluate have not shown gene mutation potential in bacteria and mammalian cells as well as in in vitro clastogenicity. Further testing is not required. For further information on the toxicity of the individual assessment entities, please refer to the relevant sections in the IUCLID and CSR.

Justification for classification or non-classification

Zinc m-toluate is not to be classified according to regulation (EC) 1272/2008 as genetic toxicant, since all in vitro and in vivo studies with the respective moieties did not show any gene mutation potential.