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In vitro Micronucleus test in Chinese hamster V79 cells

 

The in vitro Micronucleus test in Chinese hamster V79 cells with Benzylamine was conducted according OECD Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test“.

The test item Benzylamine, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. The study design was as follows:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation (S9 -mix). In Experiment II the exposure period was 24 hours without and 4 hours with metabolic activation. The cells were prepared 24 hours (Exp.I and II) after start of treatment with the test item.

In each experimental group two parallel cultures were analyzed and at least 1000 cells per culture were scored for micronuclei. The highest applied concentration (1072.0 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline No. 487.

No relevant influence on osmolarity was observed. The pH value was slightly increased at the highest applied concentration.

Neither in the absence nor in the presence of S9 mix cytotoxicity measured as reduced proliferation index was observed up to the highest required concentration.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluated concentration of 1072.0 μg/mL. The rates of micronucleated cells after treatment with the test item (0.30 - 1.50 % micronucleated cells) were close to the solvent control values (0.65 - 0.95 % micronucleated cells) and in the range of the historical solvent control data.

In Experiment II in the absence of S9 mix after 24 hours continuous treatment statistically significant increases (1.25, 2.28, 7.75 and 3.63 % micronucleated cells) were observed in the concentration range of 134.0 to 1072.0 μg/mL. At the three highest evaluated concentrations the number of micronucleated cells was determined of each test group in a sample of 4000 cells. These values clearly exceeded the historical control data range of 0.05 - 1.50 % (total: aqueous and organic solvents) (0.15 -1.5 % for aqueous solvents only) micronucleated cells.

Mitomycin C (0.3 μg/mL) (without S9 mix), Griseofulvin (8.0 μg/mL) (continous, without S9 mix) and CPA (15 μg/mL) (with S9 mix) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.

In conclusion, under the experimental conditions reported, the test item Benzylamine induced micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence of metabolic activation.

Therefore, Benzylamine has to be considered as mutagenic in this in vitro test system, when tested up to the highest required concentration of

1072.0 μg/mL.

 

 

Bacterial reverse mutation assay (Ames Test)

 

The bacterial reverse mutation assay was conducted according to the OECD guideline 471.

The test substance Benzylamine was tested for mutagenicity in the Salmonella typhimurium/ Escherichia coli reverse mutation assay both in the standard plate test (SPT) and in the preincubation test (PIT) with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The dose ranges were 33 - 5 000μg/plate for SPT and PIT.

After the bacteria were incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. As positive control 2-aminoanthracene (2-AA) (with S9 mix ) was used for all tested stains. Without S9 mix following positive controls were used:

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for strains: TA 1535, TA 100

4-nitro-o-phenylenediamine (NOPD) for strain: TA 98

9-aminoacridine (AAC) for strain: TA 1537

4-nitroquinoline-N-oxide (4-NQO) for strain: E. coli WP2 uvrA

As vehicle control ultrapure water was used.

According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and

preincubation assay). Besides, the results of the vehicle as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this method. In this study with and without S9 mix, the number of revertant colonies in the vehicle controls was within the range of the historical vehicle control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Thus, under the experimental conditions chosen here, it was concluded that Benzylamine is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

 

 

HPRT Test

 

The study was performed to investigate the potential of Benzylamine to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to the OECD guideline 476.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 1100 μg/mL was equal to approximately 10 mM (considering the preliminary information concerning the purity of the test item (≥ 99%) at the start of the experiment). The test item was dissolved in deionised water.

The tested concentrations of the main experiments ranged from 68.8 to 1100.0 μg/mL .

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Benzylamine is considered to be non-mutagenic in this HPRT assay.

 

In vivo Micronucleus test in Bone Marrow Cells of the Mouse

 

The study was performed to assess the potential of the test substance Benzylamine to induce clastogenic and spindle poison effects in NMRI mice after a single oral administration using the micronucleus test method according to the OECD guideline 474, US EPA OPPTS 870.5395 and Commission Regulation (EC) No 440/2008; B.12.

For this purpose, the test substance, dissolved in deionized water, was administered once orally to male animals at dose levels of 150 mg/kg, 300 mg/kg and 600 mg/kg bw in a volume of 10 mL/kg bw in each case. 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. A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at 600 mg/kg bw at 48-hour sacrifice interval. In this test group one animal died before reaching the intended time point for sacrifice 48 hours after test substance administration. A dose-related, statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed in the test substance treated test groups at 24-hour sacrifice interval. In addition, at 48-hour sacrifice interval the rate of micronucleated polychromatic erythrocytes was statistically significantly increased at 600 mg/kg bw. However, all values were clearly within the test facility`s historical negative control data range (0.3 – 3.2‰ micronucleated erythrocytes) and, therefore, the observation had to be regarded as biologically irrelevant. Thus, under the experimental conditions of this study, the test substance Benzylamine did not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.


Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study.

Short description of key information:
AMES Test: The test substance, Benzylamine is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
HPRT Test: In the HPRT assay the test substance, Benzylamine is considered to be non-mutagenic in the absence and the presence of metabolic activation.
MNT Test in vitro: The test substance, Benzylamine is mutagenic in the in vitro Micronucleus test in Chinese hamster V79 cells in the absence of metabolic activation.
MNT Test in vivo: The test substance Benzylamine did not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance Benzylamine was positive in the in vitro Micronucleus test in Chinese hamster V79 cells in the absence of metabolic activation, but negative in the in vivo Micronucleus test in mice. Thus, Benzylamine, is not subject to classification for mutagenicity according to the criteria of Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).