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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

gene mutation in bacteria: positive
gene mutation in mammalian cells: negative
chromosomal aberration: negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test: negative

Additional information

The genetic toxicity potential of the substance was evaluated in the in vitro gene mutation study in bacteria, in the in vitro gene mutation study in mammalian cells and in the in vitro chromosome aberration study.


 


in vitro gene mutation study in bacteria
In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 TA1537 of S. typhimurium and E.coli WP2 uvrA were exposed to the test item at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II). Test item was tested up to the limit concentration of 5000 µg/plate in all tester strains used. The positive controls induced the appropriate responses in the corresponding strains. There was evidence of induced mutant colonies over background.


 


in vitro gene mutation study in mammalian cells
In a mammalian cell gene mutation assay (HPRT locus),V79 cells cultured in vitro were exposed to tets item suspended in MEM + 0% FBS (4h treatment) and MEM + 10% FBS (20h treatment) at concentrations of:


- 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 17.5, 20.0, 22.5 and 25.0 µg/mL (without metabolic activation, Experiment I)


- 100, 150, 200, 250, 300, 400, 450 and 550 µg/mL (with metabolic activation, Experiment I)


- 5, 10, 25, 50, 75, 100, 150, 200, 250 and 300 µg/mL (without metabolic activation, Experiment II)


- 10, 20, 40, 80, 130, 160, 190, 220 and 265 µg/mL (with metabolic activation, Experiment II).


The test item was tested up to cytotoxic concentrations.


Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 14.6% for the highest concentration (25.0 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 550 µg/mL with a relative growth of 15.8%. In experiment II without metabolic activation the relative growth was 11.9% for the highest concentration (300 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 265 µg/mL with a relative growth of 8.8%.
In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.17 was found at a concentration of 1.0 µg/mL with a relative growth of 83.3%.
In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.18 was found at a concentration of 550 µg/mL with a relative growth of 15.8%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.10 was found at a concentration of 150 µg/mL with a relative growth of 32.0%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.52 was found at a concentration of 10 µg/mL with a relative growth of 104.0%.
According to OECD 476 the highest concentration tested should induce a reduced level of relative survival of approximately 10-20%. In experiment II with metabolic activation this criteria was not met due to the steep toxicity gradient and strong precipitation of the test substance. Due to the fact that in experiment I with metabolic activation all the required criteria are met and all the stages of relative survival have been covered without any hint at mutagenicity, experiment II with metabolic activation is still considered as valid.
The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background.


 


in vitro chromosome aberration study
To investigate the potential of test item to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.
The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations (except for 100 µg/mL (400 cells) and 400 µg/mL (176 cells) with metabolic activation).Based on the results of the solubility test the best suited vehicle was MEM cell culture medium (MEM + 10% FBS).
The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:
Experiment I:
without metabolic activation: 30, 60 and 100 µg/mL
with metabolic activation: 100, 200 and 400 µg/mL
Experiment II:
without metabolic activation: 20, 50 and 110 µg/mL
with metabolic activation: 100, 250 and 400 µg/mL
In experiment I and II, precipitation of the test item was seen without and with metabolic activation in all dose groups evaluated.
In experiment I without metabolic activation, cytotoxic effects of the test item were noted at a concentration of 100 µg/mL considering the relative mitotic index. However, considering the relative cell count cytotoxic effects were observed at 60 µg/mL and higher. With metabolic activation cytotoxic effects of the test item were noted at a concentration of 400 µg/mL considering the relative mitotic index and the relative cell count.
In experiment II without metabolic activation, cytotoxic effects of the test item were observed at a concentration of 110 µg/mL considering the relative mitotic index. However, considering the relative cell count no cytotoxic effects were noted at all evaluated concentrations. With metabolic activation, no cytotoxic effects of the test item were noted at all concentrations evaluated considering the relative mitotic index. Considering the relative cell count cytotoxic effects were observed at a concentration of 400 µg/mL.
In experiment I without metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. In experiment I with metabolic activation the lowest concentration displayed an increase in the aberration rate after treatment with the test item. However, this increase showed no dose-response relationship and was not reproducible in the second main experiment with metabolic activation.
In experiment II no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.
In experiment I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls.
In the second main experiment with metabolic activation at a concentration of 400 µg/ml an increase in endomitotic frequency (9% for culture 1 and 12% for culture 2) was observed. This could possibly be due to an impending effect of the test item on the spindle apparatus. However, as this effect was not observed in the first experiment it could not be verified.
EMS (400 and 600 µg/mL) and CPA (0.83 and 1.11 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.
The positive controls induced the appropriate responses. There was no evidence of test item induced over background.


 


in vivo mammalian erythrocyte micronucleus test
In a NMRI mouse peripheral blood micronucleus assay, five male and female animals per dose group were treated ip with test item at doses of 250, 125 and 50 mg/kg bw.  Peripheral blood cells were harvested at 44 h (all dose and control groups) and 68 h (negative control and 1 MTD group) post-treatment.  The vehicle was 0.9% NaCl. The animals received the test item once ip.
There were signs of toxicity during the study.  The animals treated with doses of 0.2 and 0.5 MTD showed dose-depended mild and moderate signs of systemic toxicity. The animals treated with a dose of 1 MTD showed moderate signs of systemic toxicity such as reduction of spontaneous activity, prone position, constricted abdomen, piloerection, opisthotonos, kyphosis, half eyelid closure, catalepsis and eye closure. Additionally, the animals were coloured blue. One male animal (1 MTD group) died 24 hours after treatment and was replaced by a reserve animal. Test item was tested at an adequate dose based on OECD 474. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two following categories:


- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or


- substances, which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.


 


The test substance did not show any reasons of concern in the test performed.


Therefore, the substance is not classified for genetic toxicity according to the CLP Regulation (EC) No. 1272/2008.