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In vitro data

Mutagenicity in bacterial reverse mutation assays (Ames test) has been investigated with the Amine heads category substances DCH, MPMD and HMD. Negative results were obtained in all tests with and without metabolic activation. The appropriate reference mutagenes showed distinct positive mutagenic effects.

In an Ames test similar to OECD 471 MPMD was tested on its mutagenic activity. Therefore different concentrations (0, 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate) were incubated with Salmonella typhimurium TA 1537 and E.coli WP2 uvrA. No mutagenic activity was detected with and without metabolic activation. 

In an additional Ames test Salmonella typhimurium TA 1535 TA 97, TA 98 and TA100 were used. No mutagenic activity was detected with and without metabolic activation as well. In combination these two studies can be used in a weight of evidence strategy to fulfil this endpoints requirement.

In addition a not reliable study on the mutagenic activity of DCH is available. Again no mutagenic activity was detected.

Bacterial reverse mutation tests, which were assessed to be non-reliable as one bacterial strain crucial in today’s standard methods was missing, conducted with HMD as test material revealed that this member of the amine heads category shows no mutagenic activity with or without metabolic activation system and tested in various vehicles.

Gene mutation in mammalian cells has been investigated in one reliable study (OECD 476) with MPMD (Mouse Lymphoma Assay) Negative results were obtained in the presence and absence of metabolic activation. Under the test conditions MPMDdid not induce gene mutations in concentrations up to 10 mmol/L. In another reliable study according to OECD 476 HMD was used as test material. Under the test conditions (i.e. test up to maximum of 250 µg/ml without metabolic activation and tested up to 600 µg/ml with 5% metabolic activation system), HMD was found not to induce mutations in this in vitro CHO/HGPRT mammalian cell forward gene mutation assay. In any the test the positive controls yielded the expected increase of mitant frequencies and the negative control values were within acceptable ranges.

There is one reliable in vitro study (according to OECD 473) for each category member on the induction of chromosome aberration in mammalian cells. Within these tests negative results were obtained with and without metabolic activation.

In vivo data

Using HMD-dihydrochlorid as a test material a mammalian erythrocyte micronucleus test was performed (OECD 474).A 13-week study of the toxicity of the dihydrochloride salt of HMD (HDDC) was conducted in B6C3F1 mice (both sexes) exposed by whole body inhalation in compliance with US-FDA GLP. Animals were exposed to HDDC at concentrations of 0, 1.6, 5, 16, 50 and 160 mg HDDC/m3 for 6 hours/day, 5 days/week. At the end of the 13-week inhalation study, smears were prepared from peripheral blood samples and ten thousand normochromatic erythrocytes (NCEs) and 2000 polychromatic erythrocytes (PCEs) from each animal were scored for micronuclei.

A statistically significant increase in the percentage of PCEs in the total erythrocytes population was observed for males inhaling HDDC for 13 -weeks at 50 mg/m3 and higher (not dose-related) and females exposed at 160 mg/m3, without a statistically significant increase in the frequency of micronucleated normochromatic and polychromatic erythrocytes. Under the test conditions, HMD didn't induce micronucleated erythrocytes in rats exposed by whole-body inhalation for 90 days

Moreover a mammalian bone marrow chromosome aberration test (similar to OECD 475) was performed using HMD as test material. Therefore the clastogenic potential of HMD was measured by increases in numerical and structural chromosomal aberrations in bone marrow cells from male and female Sprague-Dawley rats.

A single dose of HMD was administered by oral gavage to four groups of rats (24/sex) at levels of 0, 75, 250 and 750 mg/kg of body weight. Surviving animals from each group were sacrificed at 6, 12, 24 and 48 hours after the single administration of HMD. Under the test conditions of this in vivo cytogenicity study, HMD is not considered as clastogenic in the rat.

Short description of key information:
In vitro data for the submission substance indicate that this substance has no potential to induce gene mutations in bacteria or mammalian cells. Furthermore no cytogenic (i.e. chromosomal aberrations) were observed. Moreover various test results for other amines, which are grouped with the submission substance into the amine heads category, point to neither mutagenic nor clastogenic activities. In vivo studies (in vivo micronucelus test and in vivo chromosomal aberration test) conducted with HMD, a member of the amine heads category, support the negative in vitro findings, too.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In a reliable set of bacterial mutation assays, gene mutation studies in mammalian cells and in vitro chromosomal aberration tests as well as in vivo cytogenicity studies in rats and mice the test item is considered to be non-mutagenic and is not a clastogenic agent.

Based on the available data no classification according to Regulation (EC) No. 1272/2008 and Council Directive 67/548/EEC on mutagenicity is warranted.

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