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Additional information

Ames test:

A reverse gene mutation assay was conducted in line with Guidance for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Test Guidelines 471 and 472, using the pre-incubation method. Therefore it was identified as a key study.

In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98, TA1537 of S. typhimurium and Escherichia coli Wp2 uvrA were exposed to tetrahydromethylphthalic anhydride in DMSO at concentrations of 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate (-S9 mix) and 0, 156, 313, 625, 1250, 2500, 5000 ug/plate (+S9 mix) in the presence and absence of mammalian metabolic activation (S9 mix, Rat liver, induced with Phenobarbital and 5,6-benzoflavone).

Tetrahydromethylphthalic anhydride was tested up to limit concentration (5000 µg/plate). No cytotoxicity occurred. The test chemical did not induce mutations in the S. typhimurium and E. coli strains The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals Test Guideline and OECD 471 and 472 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Chromosome Aberration tests:

Two chromosome aberrration tests were conducted with the test substance tetrahydromethylphthalic anhydride (MTHPA).

In a first mammalian cell cytogenetics assay (supporting, Tanaka, 1997), CHL/IU cell cultures/primary lymphocyte cultures were exposed tetrahydromethylphthalic anhydride (MTHPA, CAS 11070-44-3) at concentrations of 0, 0.075, 0.15, 0.30 and 0.60 mg/ml with and without metabolic activation (S9).

Structural chromosomal aberrations were not induced up to 0.30 mg/ml (24 and 48hr continuous treatment without S9). Polyploidy (1.13 %) was increased at 0.30 mg/ml with 48 hr continuous treatment without metabolic activation. Furthermore, polyploidy (1.25-1.88 %) was statistically increased at 0.11-0.43 mg/ml (all concentrations) with short-term treatment with an exogenous metabolic activation system, indicating a potential effect of the test item. Whereas this study showed no indication of clastogenic properties, a polyploidy inducing effect cannot be excluded. Therefore a second test was performed. However, the biological relevance of the observed variation was considered equivocal as the test design is of limited use to investigate such effects.

In the key study (Beres, 2010), the test item, tetrahydromethylphthalic anhydride (MTHPA) was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation).

In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/ml test item.

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/ml test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when tetrahydromethylphthalic anhydride (MTHPA) was examined up to cytotoxic concentrations (1.22 2.44, 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml) without S9 mix over a prolonged treatment period of 20 hour and 20 and 28 hour sampling times. Further, a three-hour treatment with tetrahydromethylphthalic anhydride (MTHPA) up to cytotoxic concentrations (4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml) in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps.

As in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The validity of the test was shown using Ethylmethane sulphonate (0.4 and1.0 μl/ml) and N-Nitrosodimethylamine (1.0 μl/ml) as positive controls.

In conclusion, tetrahydromethylphthalic anhydride (MTHPA) tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells and no indications of polyploidy were noted. Therefore, tetrahydromethylphthalic anhydride (MTHPA) is considered as not clastogenic in this system. 

Mouse Lymphoma assay:

In the performed mutation assays (Vertesi, 2009), the cell cultures were treated with a range of tetrahydromethylphthalic anhydride (MTHPA) concentrations. The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments. In the Assay 1, in the absence of S9 Mix no toxic effect of the test item was observed, the changes of the harmonised relative survival and RTG values were in the ranges of the biological variability of the test system. In the presence of S9 Mix 34.22 % harmonised relative survival was observed at the highest examined concentration level, at 1800 μg/mL. The obtained mutation frequencies did not show any statistical or biological significant differences from the negative control values (Dunnett’s Test, α = 0.05). In all phases of the Assay 2 there were one or two concentration levels where the obtained mutation frequencies statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were dose-related, but the GEF criterion for positive call was not attained in any case. In the case of the 3-hour treatments (in absence of S9) the higher frequencies were observed in the repeated (partially completed) assays only. In the Assay 1 no statistical significant differences were detected between the treatment and vehicle control mutation rates. Thus, the obtained statistical significances were regarded as not biologically relevant.

REACH Regulation 1907/2006 (Annex VIII, 8.4 Column 2) states that appropriate in vivo mutagenicity studies should be considered in those cases of a positive result in any of the in vitro genotoxicity studies. In vitro investigations were negative and in vivo studies are therefore regarded as inappropriate and not in line with current concerns regarding animal welfare and the use of animals in scientific experiments.


Short description of key information:
The test substance tetrahydromethylphthalic anhydride (MTHPA) was investigated for genotoxic effects in vitro for 3 different endpoints. It was shown to be not mutagenic in the ames test with and without metabolic activation (S9). A first chromosome aberration test with CHL/IU cells revealed equivocal results. Thus a second state of the art chromosome aberration test with V79 cells was conducted. In this test MTHPA was shown to be not clastogenic. Additionally it could be shown in a mouse lymphoma assay, that MTHPA has not the potential to induce mutations in mammalian cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on results of the three different in vitro genetic toxicity studies, tetrahydromethylphthalic anhydride (MTHPA) was not classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and to Regulation 1272/2008/EC (CLP).