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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Three in vitro studies on genetic toxicity are available. All indicate that the submission item is not genotoxic.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study without deviations, performed under GLP
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster Lung (CHL) cell line
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Experiment I, without S9: 3.75, 7.5, 15, 30, 45, 60 µg/mL
Experiment I, with S9: 15, 30, 45, 60, 90, 120 µg/mL

Experiment II, 24-hour exposure, without S9: 0.31, 0.63, 1.25, 2.5, 3.75, 5 µg/L
Experiment II, 48-hour exposure, without S9: 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 µg/L
Experiment II, 6-hour exposure, with S9: 7.5, 15, 30, 45, 60, 75 µg/L
Vehicle / solvent:
Dimethyl sulfoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I, with and without S9: 6 hours
Experiment II, without S9: 24 and 48 hours
Experiment II, with S9: 6 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment I, with and without S9: 24 hours
Experiment II, without S9: 24 and 48 hours
Experiment II, with S9: 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL (2 hours prior to harvest time)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS
guidelines for mutagenicity testing. Evaluation of metaphase cells may be terminated after 50 cells if approximately 50% cells with aberrations were observed.
Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells are recorded and are included in the polyploid cell total number.
If there was a dose-related increase in endoreduplicated cells then they are reported separately. The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. The number of gap-type aberrations was recorded and reported.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. If the study gives a positive response then a D20 value will be calculated, which is the presumed dose level of the test substance that is required to induce aberrations in 20% of metaphases.
Species / strain:
other: Chinese Hamster Lung (CHL) cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: no metaphase cells present at >=30 µg/mL (without S9) and 90 µg/mL (with S9). Experiment II: 50% growth inhibition at >=2.5 µg/mL (24-hour group) and >=0.25 µg/mL (48-hour group), 39% cell growth inhibition at 75 µg/mL (6 hours group)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the Cell Growth Inhibition test, a precipitate of the test material was seen at and above 1500 µg/mL. In the main test, precipitate of the test material was not observed at the end of the treatment period in any exposure group.

RANGE-FINDING/SCREENING STUDIES: A Cell Growth Inhibition test was performed. In all cases the test material showed evidence of cell toxicity. A precipitate of the test material was seen at and above 1500 µg/mL. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphases were present at dose levels up to 50 µg/mL in the 6(18)-hour with-S9 exposure group and at up to 15 µg/mL in the 6(18)-hour without-S9 exposure group. The maximum dose with metaphases present in the 24-hour continuous exposure was 10 µg/mL and 2.5 µg/mL in the 48-hour exposure group. The dose selection for the main experiments was based on toxicity for all exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA: The data lay within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I, the test material demonstrated similar toxicity to that observed in the Cell Growth Inhibition Test. These data show an approximate 50% growth inhibition was not achieved in the absence or presence of S9. The test material was shown to have a very steep toxicity curve, such that there were no metaphase cells present at and above 30 µg/mL in the absence of S9 and 90 µg/mLl in the presence of S9. It was considered that adequate levels of toxicity had been achieved.
In Experiment II, the test material demonstrated similar toxicity to that observed in the Cell Growth Inhibition Test. The data show that an approximate 50% growth inhibition was achieved at 2.5 µg/mL and above in the 24-hour exposure group and at 0.25 µg/mL and above in the 48-hour exposure group. In the 6(18)-hour exposure group in the presence of S9 39% cell growth inhibition was achieved at the maximum dose level tested of 75 µg/mL. In a previous experiment this same dose level had proved to be too toxic for evaluation. It was therefore considered that the test material had been adequately tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment II

The test material induced small but statistically significant increases in the frequency of cells with aberrations in the 48 hour continuous exposure group and in the 6(18)-hour exposure group. It should be noted however, that in both cases the responses were within the historical maxima for that time point, both were in comparison to very low vehicle control values and were not part of any dose-related response. Therefore, both increases were considered to be of no toxicological significance.

Polyploid cells

The test material did not induce any statistically significant increases in the number of polyploid cells at any dose level in either treatment case.

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce any toxicologically significant, dose-related increases in the frequency of cells with chromosome aberrations, either in the presence or absence of a liver enzyme metabolising system, or after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.
Executive summary:

This study was conducted according to a method which was designed to assess the potential chromosomal mutagenicity of a test material on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to OECD TG 473.

Duplicate cultures of Chinese Hamster Lung (CHI+) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure, a 48-hour continuous exposure and a repeat of the 6(18)-hour exposure with metabolic activation.

The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 3.75 to 120 µg/mL for the 6(18)-hour exposure, both with and without S9, and 0.25 to 5 µg/mL for the 24 and 48-hour treatments.

The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The test material was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved.

The test material was shown to be non-clastogenic to CHL cells in vitro.

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

Additional information

Additional information from genetic toxicity in vitro:

Mutagenic potential

This GLP study was designed to assess the mutagenic potential of the test material. The method meets the requirements of the OECD TG 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA¯ were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding study was determined in a preliminary toxicity assay and ranged between 0.5 and 1500 µg/plate depending on presence or absence of S9-mix. The experiment was repeated on a separate day using a similar dose range to the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were included in both experiments to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the bacterial tester strains, initially at 150 and 500 µg/plate without and with S9, respectively. The test material was, therefore, tested up to the toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

Chromosomal Mutagenicity

The potential chromosomal mutagenicity of a test material on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line was assessed according to OECD TG 473. Duplicate cultures of Chinese Hamster Lung (CHI+) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure, a 48-hour continuous exposure and a repeat of the 6(18)-hour exposure with metabolic activation.

The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 3.75 to 120 µg/mL for the 6(18)-hour exposure, both with and without S9, and 0.25 to 5 µg/mL for the 24 and 48-hour treatments.

The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The test material was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved.

The test material was shown to be non-clastogenic to CHL cells in vitro.

Potential to induce mutations

The study was performed to investigate the potential of Gaskamine 240 to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The study was performed under GLP and followed OECD TG 476.

The study was performed in three independent experiments with and without liver microsomal activation. The highest concentration (3440 μg/mL) applied in the pre-experiments was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects of the test item. Experiment I was performed with and without metabolic activation with a treatment time of 4 hours. Experiment II was performed with a treatment time of 24 hours in the absence and 4 hours in the presence of metabolic activation.

Relevant cytotoxic effects indicated by a relative total growth (RTG) of less than 50% of survival in both parallel cultures were observed in the first experiment at 10.0 and 20.0 μg/mL with and at 5.0 μg/mL without metabolic activation. In the second experiment cytotoxic effects as described above were noted at 10.0 μg/mL with and at 3.8 and 5.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative total growth was covered in the second experiment with and in the first experiment without metabolic activation. In the first experiment with and the second experiment without metabolic activation the RTG was in a range from 5.5 to 8.0% at the maximum analysable concentration. Although the RTG dropped below 10% the data are acceptable since the exception criteria set by the IWGT apply. According to those criteria a set of data is acceptable provided that there is no evidence of mutagenicity in a series of data points between 100% to 25% and there is also a negative data point between 10% and 1% RTG.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. In the first culture of the first experiment without metabolic activation the threshold of 126 plus the solvent control count and the historical range of solvent controls was exceeded at 2.5 μg/mL. This isolated increase was judged as irrelevant since no comparable increase was noted in the parallel culture under identical conditions or at the next higher concentration in both parallel cultures. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. Another irrelevant increase of the mutation frequency exceeding the threshold mentioned above occurred in the second culture of the second experiment with metabolic activation at 10 μg/mL. This increase, however, is based on the low solvent control. The absolute value of the mutation frequency remained within the range of the historical solvent controls.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Gaskamine 240 is considered to be non-mutagenic in this mouse lymphoma assay.

Conclusion

All the tests on genotoxicity were negative. The test item is therefore considered to be non-genotoxic.


Justification for selection of genetic toxicity endpoint
Klimisch 1, guideline-conform study without deviations, performed under GLP.

Justification for classification or non-classification

The test item is considered to be non-genotoxic, no classification therefore applicable.