Barium m-toluate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No genetic toxicity study with barium m-toluate is available, thus the genetic toxicity will be addressed with existing data on the individual moieties barium and m-toluate. Since none of the in vitro genotoxicity studies rated as reliable for the moiety barium showed any effect in bacterial reverse mutation assays, in mammalian cell gene mutation tests (TK assay) or in mammalian cell chromosome aberration tests, an in vivo follow-up of genotoxicity was not required for that assessment entity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Barium

None of the in vitro genotoxicity studies rated as reliable showed any effect in bacterial reverse mutation assays, in mammalian cell gene mutation tests (TK assay) or in mammalian cell chromosome aberration tests, thus the classification criteria according to regulation (EC) 1272/2008 as germ cell mutagen are not met.

 

in vitro clastogenicity

Based on the outcome of guideline-compliant studies barium dichloride does not induce chromosome aberrations in mammalian cells, when tested up to toxic and/or precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9 mix).

Overall it can be concluded that barium dichloride does not induce chromosome aberrations in vitro in somatic mammalian cells. Therefore the conduct of in vivo clastogenicity experiments is not required.

  

in vitro gene mutation

Anonymous (1994)

The authors state that barium dichloride induces gene mutations in cultured mouse lymphoma cells (L5178Y) in the presence of S9 in a statistical significant manner. However, the mutation frequency increased from 32 per 106

cells in the control culture to a maximum of 59 per 106 cells at 1000µg/mL (with a RTG of 10%). Being a statistical significant increase in mutation frequency, the biological significance however is considered questionable, since the highest MF is still well below the value recommended by the IWGT (Moore et al., 2003; Moore et al., 2006; Moore et al., 2007) of 154 per 106 cells. Furthermore, a comparison with historical data for the performing laboratory is not possible, since the data was not given in the study report.

Due to the questionable biological relevance, the statistical significant increase in mutation frequency in both barium dichloride cultures with metabolic activation is not considered as clear positive response. Therefore, it was decided to repeat the whole experiment under clearly defined conditions, which a highly pure test item under guideline and GLP compliant conditions.

 

Lloyd (2010)

It is concluded that barium dichloride did not induce gene mutations in the TK locus of L5178Y mouse lymphoma cells when tested up to toxic and/or precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9 mix).

Overall it can be concluded that barium dichloride does not induce gene mutations in vitro in bacteria and somatic mammalian cells. Therefore, the conduct of in vivo gene mutation experiments is not required.

 

References

Moore M et al. (2003)

Mouse lymphoma thimidine kinase gene mutation assay: International workshop on Genotoxicity tests workgroup report – Plymouth, UK 2002. Mutation Research (2003), 540, 127-140.

 

Moore M M, Honma M, Clements J, Bolcsfoldi G, Burlinson B et al. (2006)

Mouse lymphoma thymidine kinase gene mutation assay: follow up meeting of the International Workshop on Genotoxicity Testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and date evaluation. Environmental and Molecular Mutagenesis 2006, 47, 1-5.

 

Moore M M, Honma M, Clements J, Bolcsfoldi G, Burlinson B et al. (2007)

Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment. Mutation Research 2007, 627, 36-40.

 

 

m-toluate

Reliable data on the mutagenicity of m-toluic acid are available from bacterial and mammalian cells gene mutation assays and an in vivo micronucleus tests in rats. m-Toluic acid showed in none of these tests a genotoxic potential.

 

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. 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 bw 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 bw. 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 bw. However, this finding was not reproduced in a second confirmation test, in which five animals were exposed to 125, 250, and 500 mg/kg bw 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.

Barium m-toluate

Barium m-toluate is not expected to be genotoxic, since the two moieties barium and m-toluic acid have not shown gene mutation potential in bacteria and mammalian cells as well as in vitro clastogenicity. Further testing is not required. Thus, barium m-toluate is not to be classified according to regulation (EC) 1272/2008 as genetic toxicant. For further information on the toxicity of the individual constituents, please refer to the relevant sections in the IUCLID and CSR.

Justification for classification or non-classification

As the two moieties of barium m-toluate do not induce genotoxic effects leading to a classification, barium m-toluate in all probability has also no potential to induce genotoxic effects.

According to the criteria of REGULATION (EC) No 1272/2008 and its subsequent adaptiations, barium m-toluate does not have to be classified and has no obligatory labelling requirement for germ cell mutagenicity.