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Key value for chemical safety assessment

Additional information

In vivo

 

Micronucleus test

The test substance was tested in a mouse micronucleus assay according OECD guideline no. 474 and EU method B.12. Possible clastogenic effect to the chromosomes of bone-marrow erythroblasts of the test substance were investigated in male and female mice.

The treated animals received a single intraperitoneal administration of either test substance or cyclophosphamide (positive control). The femoral marrow of groups treated with test substance was prepared 16,24 and 48 hours after administration. All negative and positive control animals were sacrified after 24 hours. The doses of test substance and positive control were 15 and 20 mg/kg bw, respectively. The animals treated with test substance showed symptoms of toxicity after administration. One of the forty animals died before the end of the test due to the acute inraperitoneal toxicity of 15 mg/kg test substance. There was weekly altered ratio between polychromatic and normochromatic erythrocytes.

The results with the test substance gave no relevant indications of clastogenic effects after single intraperitoneal treatment with 15 mg/kg. The known mutagen and clastogen, cyclophosphamide, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg bw. The number of micronucleated polychromatic erythrocytes increased to a biologically relevant degree.

The number of microncleated normochromatic erythrocytes did not increase relevantly in any of the groups. The test substance was judged to be not clastogenic in vivo.

In vivo alkaline comet assay

The purpose of the comet assay (single cell gel electrophoresis assay) was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs, tissues. The genotoxicity potential of the test item was investigated on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day on day 0 and 24 hours thereafter. The negative control (propylene glycol 400) and positive controls (1,2-dimethylhydrazine dihydrochloride and ethyl methanesulfonate) were tested in parallel. Each dose group and the negative control group consisted of 6 animals; the positive control groups consisted of 4 animals.

Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 200, 100 and 50 mg/mL. The measured concentration values remained within the ± 5% of nominal range at 200 and 100 mg/mL concentrations (99 and 100 % for the 200 mg/mL solution; 95 and 96 % for the 100 mg/mL solution); but slightly lower values 87 % of the nominal were obtained at the nominal concentration of 50 mg/mL. In this particular unequivocally negative test the somewhat lower measured concentration levels (especially the measured: 43.5 and 43.7 mg/mL instead of 50 mg/mL (435 and 437 mg/kg bw/d instead of 500 mg/kg bw/d) have not any influential effect on the results and final conclusion of the study; therefore the nominal concentration values 200, 100 and 50 mg/mL (2000, 1000 and 500 mg/kg bw/d) were applied and referred throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method (Study code: 644-100-4845).

3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive controls) the animals were euthanized and the cells of the target tissues were isolated.

Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling.

The slide preparation was done within one hour after single cell preparation. Therefore the following steps were performed: embedding the cells, lysis (pH > 10), alkaline unwinding (pH > 13) followed by electrophoresis (pH > 13). After electrophoresis slides were neutralized and preserved in absolute ethanol. Just prior scoring the DNA, the slides were stained using Ethidium bromide.

For each animal, each tissue 4 slides were prepared (8 slides per animal, 48 slides per test item doses and negative control and 32 slides per positive controls).

Three slides of five animals per vehicle control and test item treatments were stained and analysed (6 slides per animal, 30 slides per dose) and three slides of three animals per positive controls were stained and analysed (6 slides per animal, 18 slides per dose).

The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) was used.

For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive controls).

In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection.

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met (See: Validity of the Study).

No mortality was observed during the treatments and expression period in any dose group and in the controls. Toxic symptoms any clinical signs were not observed during the treatments. At the tissue isolation after the opening of the stomach a characteristic strong chemical smell was noticed at the test item treatments and the smell intensity increased dose-dependently.

The animal weights increased in all treatments, test item, negative and positive control treatments. The weight increases remained in the same range 3.56-4.07 % at the negative and positive controls and at the doses of 500 and 1000 mg/kg bw/d and the increase was lower 2.28 % at the highest dose group of 2000 mg/kg bw/d.

At the screening cytotoxicity measurements (using Trypan blue dye exclusion method) significant cytotoxicity was not noticed in any test item and control item treatments.

The number of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the positive control treatments in the stomach samples.

The numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and at the EMS positive control treatments in the liver samples, and a statistically significant increase of ghost cells was noticed at the DMH treatments in the case of liver samples.

DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, Olive Tail Moment (OTM) and tail length.

The % tail DNA mean values of each treatment were slightly lower than the vehicle control value at both, at the stomach and liver samples.

The % tail DNA mean values in the stomach samples did not differ statistically significantly from that of the vehicle control at the 500 and 1000 mg/kg bw/d doses and statistical significant difference was noticed at the 2000 mg/kg bw/d dose. The statistical significance was considered as not relevant in mutagenicity point of view since the significance linked with a lower (but acceptable) value than the corresponding vehicle control value.

The % tail DNA values in the liver samples did not differ statistically significantly from that of the vehicle control at the whole examined dose range.

Similarly to the % tail DNA values in the stomach and liver samples sporadic statistical significances occurred at the tail moment and tail length comparisons as well; however the statistical significances were not considered as biologically relevant.

Additionally the % tail DNA mean median values were investigated and found the same tendencies as at the mean values in the case of stomach samples and the mean median values of the liver samples were nearly in the same range in the vehicle control and treated doses. Any tendency and/or statistical significance at the test item treated doses was not noticed.

In conclusion, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells. The test item is negative, did not show genotoxic activity in the examined tissues.

In vitro

 

Ames test

The test substance was investigated in the Salmonella / microsome test for point-muagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 200 µg per plate did not cause any bateriotoxic effects: Total bacteria counts remained unchanged and noinhibition of growth was observed.

At higher doses, the substance had a strain-specific bacteriotoxic effect, so that this range could only be used to a limited extend up to 5000 mg per plate for assessment purposes. Evidence of mutagenic activity for the test substance was not seen. No biologically-relevant increase of the mutant count, in comparision with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene-diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically-relevant increase in mutant colonies compared to the corresponding negative controls. This result is supported by a second Ames test available. No evidence of mutagenic activity was found for the test substance. Neither a dose related doubling nor a biologically relevant increase of the mutant count in comparison with the negative controls were observed.

 

HPRT test

The test item, Incozol EH was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations and basis of solubility of test item made in a preliminary study (without and with metabolic activation using S9 mix). In the performed Main Mutation Assays the concentration levels were chosen mainly based on the solubility of test item.

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

200, 250, 300, 350, 400, 450 and 500μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

200, 250, 300, 350, 400, 450 and 500μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

200, 250, 300, 350, 400, 450, 500 and 550μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

200, 250, 300, 350, 400, 450 and 500μg/mL

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested without metabolic activation. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 1, the mutant frequency of the cells showed statistically and biologically significant increases in mutation frequency at the concentrations of 350, 400, 450 and 500μg/mL, when the test item was tested with S9-mix.

In Experiment 2, the mutant frequency of the cells showed statistically significant increases in mutation frequency at the concentrations of 400, 450 and 500μg/mL when the test item was tested with S9-mix. These increases were biologically significant and dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours), further indicating that the findings in Experiment 1 were in the normal biological variation observed, when the test item was examined in the absence of metabolic activation.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Incozol EH tested without metabolic activation (S9 mix) over a 5-hour and 20-hour period did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Incozol EH tested with metabolic activation (S9 mix) over a 5-hour treatment period induced statistically and biologically significant increases in the mutant frequency.

Incozol EH was mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

 

Conclusion

There are two bacterial reverse mutation assays (Ames test), one mammalian cell gene mutation test in vitro, one in vivo micronucleus test and one in vivo comet assay. Although the test item induced an increase of mutant frequency in mammalian cells in vitro in the presence of metabolic activation, no mutagenic activity was observed in two bacterial reverse mutation tests on Salmonella typhimurium. Additonally, there was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg bw test substance in the microncleus test on the mouse, i.e. in a somatic test system in vivo. Furthermore, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells in an in vivo Comet assay. Thus, the test substance is considered to be not mutagenic.

 


Justification for selection of genetic toxicity endpoint
Two bacterial reverse mutation assays (Ames tests), one mammalian cell gene mutation assay, one in vivo micronucleus test and one in vivo comet assay are available with the test item.

Short description of key information:
The test item induced an increase of mutant frequency in mammalian cells in vitro in the presence of metabolic activation. However, no mutagenic activity was observed in two bacterial reverse mutation tests on Salmonella typhimurium. Additionally, there was no indication of a clastogenic effect of an intraperitoneal dose of 15 mg/kg bw test substance in the microncleus test on the mouse, i.e. in a somatic test system in vivo. Furthermore, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or stomach cells in an in vivo Comet assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo the test item is considered to be not mutagenic according to Regulation (EC) No 1272/2008 (CLP).