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Additional information

There are reliable in vitro studies available to assess the potential of the test substance for both gene mutations in bacteria and cytogenicity in mammalian cells.

Gene mutation in bacteria

In a GLP conform Ames test according to OECD guideline 471, the substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate (purity: 93.0 weight-%) 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 TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA (BASF 2006). Two independent assays were performed, a standard plate test (SPT) and a preincubation test (PIT), both with a dose range of 56 µg - 5500 µg/plate, and both with and without metabolic activation (Aroclor 1254-induced rat liver S-9 mix).

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Precipitation of the test substance was found from about 557 mg/plate onward. A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions at doses >= 1750 µg/plate.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Gene mutation in mammalian cells

In an in vitro gene mutation test (HPRT) in CHO cells the substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate was assessed for its potential to induce gene mutations. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). The following concentrations were assessed:

1st experiment

without S9 mix (4-hour exposure period)

0; 6.3;12.5; 25.0; 50.0; 75.0; 100.0; 150.0; 200.0 μg/mL

with S9 mix (4-hour exposure period)

0; 31.3; 62.5; 125.0; 500.0; 750.0; 1 000.0; 1 250.0; 1 500.0 μg/mL

2nd experiment

without S9 mix (24-hour exposure period)

0; 1.6; 3.1; 6.3; 12.5; 20.0; 30.0; 40.0; 60.0 μg/mL

with S9 mix (4-hour exposure period)

0; 31.3; 62.5; 125.0; 500.0; 750.0; 1 000.0; 1 250.0; 1 500.0 μg/mL

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

In the absence of metabolic activation, in both experiments the highest concentrations applied could not be evaluated for gene mutations due to pronounced cytotoxicity. In the presence of metabolic activation, the highest concentration that could be formulated in the vehicle DMSO (1500 μg/mL) led to slightly reduced cloning efficiencies of about 50% survival in both experiments.

On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies neither without S9 mix nor after adding a metabolizing system in two experiments performed independently of each other, and apply clear cytotoxic or maximum soluble concentrations.

Thus, under the experimental conditions of this study, 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulphate is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Cytogenicity in mammalian cells

In a GLP conform study according to OECD guideline 473, the substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the presence and in the absence of a metabolizing system.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested (the test groups in brackets were not evaluated):

- 1st experiment:

4-hour exposure, 18-hour sampling time, without S-9 mix: 0; 62.5 ; 125.0; 250.0 (500.0; 750.0; 1000) µg/mL;

4-hour exposure, 18-hour sampling time, with S-9 mix: 0; (250.0;) 500.0 ; (750.0;) 1000.0; (1250.0;) 1500.0 µg/mL.

- 2nd experiment

18-hour exposure, 18-hour sampling time, without S-9 mix: 0; 6.25; 12.5; 25.0; (50.0; 75.0; 100.0; 200.0) mg/mL

For confirmation of the results of the 1st and 2nd experiments including continuous exposure and a second sampling time, the following doses were tested in the amendment report:

- 3rd experiment

18-hour exposure, 28-hour sampling time, without S-9 mix: 0; (6.3; 12.5; 25;) 50(; 100) μg/mL

4-hour exposure, 28-hour sampling time, with S-9 mix: 0; 31.3; 62.5; (125;) 500; 1000(; 1500) μg/mL

About 2-3 hours prior to harvesting the cells, Colcemid was added to arrest cells at a metaphase-like stage of mitosis (c-metaphases). After preparation of the chromosomes and staining with Giemsa, 100 metaphases for each culture in the case of the test substance, and vehicle controls, or 50 cells for each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations.

The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations.

On the basis of the results of the present study, the test substance did not cause any increase in the number of structurally aberrant metaphases incl . and excl. gaps either without S-9 mix or after adding a metabolizing system in two experiments performed ndependently of each other and apply clear cytotoxic concentrations. No increase in the frequency of polyploid cells was demonstrated either. Thus, under the experimental conditions of this assay, the test substance is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.


Short description of key information:
In vitro:
The test substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate showed no mutagenic effects in a reverse mutation test (Ames test) in bacteria (S. typhimurium/ E.coli) with and without metabolic activation.

Gene mutation in mammalian cells
The test substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate showed no mutagenic effects in an in vitro gene mutation test (HPRT) in CHO cells with and without metabolic activation.

Cytogenicity in mammalian cells
The test substance 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate showed no mutagenic effects in a chromosome aberration test in V79 cells with and without metabolic activation.

In vivo
No data available and required.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro studies, there is no indication for a mutagenic potential of the test substance. Thus, the test substance has not to be classified with regard to mutagenicity according to 67/548/EEC and to Regulation (EC) No 127272008 (GHS, CLP).