Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl]-2-oxoethyl]-4-methyl-, chloride (1:1)

Endpoint summary

Additional information

Genetic toxicity in vitro

Ames-Test

The test substance Tinocat ES 96000 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay in a study according to OECD 471 guideline and GLP.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5 000 μg/plate (SPT) 10 μg - 3 333 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 1 000 μg/plate.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Thus, under the experimental conditions of this study, the test substance Tinocat ES 96000 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT-Test

The substance Tinocat ES 96000 was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). The study was conducted according to OECD 476 guideline and GLP (BASF SE, 2013).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested. Test groups printed in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period)

0; 187.5; 375.0; 750.0; 1 500.0; 3 000.0 μg/mL

with S9 mix (4-hour exposure period)

0; 187.5; 375.0; 750.0; 1 500.0; 3 000.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period)

0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL

with S9 mix (4-hour exposure period)

0; 250; 500; 1 000; 2 000; 3 000 μg/mL

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6 - thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study, after an exposure period of 4 hours in the 1st Experiment in the presence of metabolic activation and after 24 hours exposure in the 2nd Experiment in the absence of metabolic activation strong cytotoxicity was observed at the highest required concentration tested for gene mutations. However, in all additional experimental parts toxic effects over a broad concentration range were observed as well.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance Tinocat ES 96000 is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

in vitro micronucleus assay

The substance Tinocat ES 96000 was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). A single experiment was carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). The study was conducted according to OECD 487 guideline and GLP (BASF, 2013).

Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 46.9; 93.8; 187.5; 375; 750; 1 500; 3 000 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 46.9; 93.8; 187.5; 375; 750; 1 500; 3 000 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e.2 000 cells for each test group.

The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) or proliferation index (PI) was observed at least at the highest applied test substance concentrations in both experimental parts of this study. Besides, a dose-related decrease in cell numbers was measured over a broad concentration range of about 1.5 decades.

On the basis of the results of the present study, the test substance caused a clear, statistically significant and biologically relevant increase in the number of cells containing micronuclei in the absence and presence of metabolic activation.

Thus, under the experimental conditions described, Tinocat ES 96000 is considered to have a chromosome-damaging (clastogenic) effect and/or to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

in vivo Micronucleus Test

The substance Tinocat ES 96000 was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in deionized water, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

As vehicle control, male mice were administered merely the vehicle, deionized water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range.

Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

No relevant inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the present study, the single oral administration of Tinocat ES 96000 did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

The rates of micronuclei were within the range of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

Thus, under the experimental conditions of this study, the test substance Tinocat ES 96000 does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Short description of key information:
Ames-Test: negative (BASF SE, 2011)
HPRT-Test: negative (BASF SE, 2013)
in vivo Micronucleus Test: negative (BASF, 2013)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, classification is not warranted for genetic toxicity in accordance with EU Directive 67/548 and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.