Registration Dossier
Registration Dossier
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EC number: 939-039-1 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The available data from three in vitro
assays show that the substance does not have a genotoxic potential.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 4 august 2009 - 26 August 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- other: see table 7.6.1/1
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction prepared from SD derived rats, dosed with phenobarbital and 5,6-benzoflavone, purchased from a commercial source (Moltox; Lot No. 2416).
- Test concentrations with justification for top dose:
- Test 1 and 2: 5; 15; 50; 150; 500; 1500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item soluble in dimethyl sulphoxide (DMSO, ACS spectrophotometric grade) - Untreated negative controls:
- yes
- Remarks:
- plates without bacteria to assess the sterility of the test substance, S9 mix and PBS
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- see Table 7.6.1/2
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- Remarks:
- Without S9 mix
- Untreated negative controls:
- no
- Remarks:
- plates without bacteria to assess the sterility of the test substance, S9 mix and PBS
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- see Table 7.6.1/2
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) in the first experiment and in the second experiment with S9 mix; preincubation in the second experiment withour S9 mix (30 min at 37°C)
DURATION
- Preincubation period: 30 min
- Exposure duration: 72 hours approximately
NUMBER OF REPLICATIONS: 3 plates/concentrations, 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. - Evaluation criteria:
- Acceptance criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL.
Criteria for assessing mutagenic potential:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed. If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. - Statistics:
- none
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- see tables 7.6.1/3 to 7.6.1/6
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitate observed on all plates containing the test item at 1500 and 5000 µg/plate.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: nodata
- Evaporation from medium: none
- Water solubility: not soluble in water but in DMSO
- Precipitation: in both experiments, precipitate observed on all plates containing the test item at 1500 and 5000 µg/plate.
RANGE-FINDING/SCREENING STUDIES: not applicable
COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the vehicle controls were within or close to the 99%
confidence limits of the current historical control range of the laboratory. - Conclusions:
- The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and E.coli WP2, either in the presence or in the absence of a rat liver metabolizing system.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 and EC No.B13/14 guidelines, and in compliance with the GLP, Salmonella typhimurium strains TA1535, TA1537, TA100, TA98 and E. Col WP2 were exposed to the test item diluted in DMSO at concentrations of 5; 15; 50; 150; 500; 1500 and 5000 µg/plate (triplicates) in the presence and absence of mammalian metabolic activation (fraction of S9from the liver of rats treated with phenobarbital and 5,6-benzoflavone). Both methods of direct incorporation or with a preincubation step were tested during two independent experiments. The negative control was the vehicle DMSO. Positive controls were used for each strain with and without metabolic activation and induced the appropriate responses in the corresponding strains.
No cytotoxicity was observed in any experiments. Precipitate was observed in all strains in both experiments from 1500 µg/plate. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to 5000 µg/plate in either the presence or absence of S9 mix.
The test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli as there was no evidence of induced mutant colonies over background.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 February 2016 - 05 April 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- yes
- Remarks:
- : see chapter 'any other information on materials and methods'
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine Kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
- Test concentrations with justification for top dose:
- Experiments without S9 mix: The selected dose-levels were 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the 3- and 24-hour treatments.
Experiments with S9 mix: The selected dose-levels were as follows:- 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the first experiment,- 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the second experiment. - Vehicle / solvent:
- - Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: allowed solubilization of the test item - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
- Short-term treatment: With and without S9-mix: 3 h
- Prolonged treatment period: Without S9-mix: 24 h
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days
SELECTION AGENT (mutation assays): 4 µg/mL trifluorothymidine (TFT)
NUMBER OF REPLICATIONS:
- Preliminary test: Single culture per test concentration
- Main test: Duplicate cultures per test concentration and per vehicle and positive controls
NUMBER OF CELLS EVALUATED: 2 x 10E5 cells/mL (for preliminary and main tests)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth. - Evaluation criteria:
- Positive result defined as:
- At least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 E-6.
- A dose-related trend is demonstrated by a statistically significant trend test.
Unless clearly positive, the reproducibility should be confirmed.
Negative results defined as:
- No evidence of mutagenicity at concentrations inducing moderate cytotoxicity (10% < Adj. RTG <20%),
or
If there are no cultures with moderate cytotoxicity (ie 10% < Adj. RTG <20%)
then
- at least one negative data point between 20% and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG
or
- at least one negative data point between 1% and 10% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 25% Adj. RTG - Statistics:
- To assess the dose-response relationship, a linear regression was performed between dose-levels and individual mutation frequencies obtained from cultures showing a mean Adj. RTG = 10%.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none. At the dose-level of 1000 µg/mL, the pH of the culture medium was approximately 7.4 as for the vehicle control.
- Effects of osmolality: none. At the dose-level of 1000 µg/mL, the osmolality was equal to 431 mOsm/kg H2O compared to 448 mOsm/kg H2O for the vehicle control)
RANGE-FINDING/SCREENING STUDIES:
-Based on available solubility data, the dose-level of 1000 µg/mL was expected to produce precipitation in the culture medium. Thus, the dose-levels selected for the treatments of the preliminary test were 2, 20, 100, 200, 500 and 1000 µg/mL.
- Precipitate was observed at concentrations of 100 µg/mL and greater both in the absence and presence of S9 mix following 3 and 24-hour exposure.
- Following the 3-hour treatment without S9 mix, as well as the 3-hour treatment with S9 mix, no cytotoxicity was induced at any dose-levels, as shown by the absence of decrease in the Adj. RTG.
- Following the 24-hour treatment without S9 mix, a moderate to marked cytotoxicity was induced at dose levels of 100 µg/mL or greater, as shown by a 45 to 88% decrease in the Adj. RTG.
MAIN MUTATION TEST
3 h treatment in the absence of S9 mix (cultures were exposed to the test substance at concentrations from 6.25 to 200 µg/mL):
- Precipitation was observed at concentrations of 50 µg/mL and greater. Cultures exposed to the test substance at concentrations from 6.25
to 200 µg/mL were assessed for determination of IMF.
- No cytotoxicity was induced at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the Adj. RTG.
- No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control and no dose-response relationship was demonstrated by a statistically significant trend test. These results met the criteria of a negative response.
- The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency.
3 hour treatment in the presence of S9 mix (cultures were exposed to the test substance at concentrations from 6.25 to 200 µg/mL in the first experiment and from 3.13 to 100 µg/mL in the second experiment):
- Precipitation was observed at concentrations of 50 µg/mL and greater. Cultures exposed to the test substance at concentrations from 3.13 to 200 µg/mL were assessed for determination of IMF.
- No cytotoxicity was noted as shown by the absence of any noteworthy decrease in the Adj. RTG
- No noteworthy increase in the mutation frequency was noted at any dose-levels in the first experiment and no dose-response relationship was demonstrated by a statistically significant trend test. In the second experiment (Table 9), a very slight increase in the MF was observed at the dose-level of 100 µg/mL and a dose-response relationship was demonstrated by the statistically significant trend test (p < 0.05). Nevertheless, the corresponding IMF (34 x 10-6) remained substantially below the GEF of +126 x 10-6, consequently, these results did not meet the criteria of a positive response. Since no similar increase in the MF was noted in the first experiment performed under the same experimental conditions, even up to a higher dose-level of 200 µg/mL, it was considered not to be biologically relevant. Consequently, the overall results met the criteria of a negative response.
- The positive control, cyclophosphamide, induced an acceptable increase in mutation frequency.
24 h treatment in the absence of S9 mix (cultures were exposed to the test substance at concentrations from 6.25 to 200 µg/mL.)
- Precipitation was observed at concentrations of 25 µg/mL and greater. Cultures exposed to the test substance at concentrations from 6.25
to 200 µg/mL were assessed for determination of IMF.
- No cytotoxicity was induced at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the Adj. RTG.
- No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control and no dose-response relationship was demonstrated by a statistically significant trend test. These results met the criteria of a negative response.
- The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency. - Conclusions:
- The test item, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
- Executive summary:
The objective of this study was to evaluate the potential of the test item, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells. The study was performed according to OECD 490 guideline and in compliance with the principles of Good Laboratory Practice.
After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide, was tested in two independent experiments, with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction of rats induced with Aroclor 1254.
Cultures of 20 mL at 5 x 105 cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).
The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.
With one exception which was considered not to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.
In the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.The study was therefore considered to be valid.
Since the test item was found poorly soluble in the final treatment medium during the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of precipitate, according to the criteria specified in the international guidelines.
Experiments without S9 mix
The selected dose-levels were 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the 3- and 24-hour treatments.
A precipitate was observed in the culture medium at dose-levels = 50 µg/mL at the end of the 3-hour treatment and = 25 µg/mL at the end of the 24-hour treatment.
No cytotoxicity was induced at any of the tested dose-levels, following either the 3- or the 24-hour treatment, as shown by the absence of any noteworthy decrease in the Adj. RTG.
No noteworthy increase in the mutation frequency was noted at any dose-levels, following either the 3- or 24-hour treatment,and no dose-response relationship was demonstrated by a statistically significant trend test. These results met the criteria of a negative response.
Experiments with S9 mix
The selected dose-levels were as follows:
- 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the first experiment,
- 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the second experiment.
At the end of the treatment period, a precipitate was observed in the culture medium at dose-levels = 50 µg/mL.
No cytotoxicity was induced at any of the tested dose-levels in either experiment, as shown by the absence of any noteworthy decrease in the Adj. RTG.
No noteworthy increase in the mutation frequency was noted at any dose-levels in the first experimentand no dose-response relationship was demonstrated by a statistically significant trend test. In the second experiment, a very slight increase in the MF was observed at the dose-level of 100 µg/mL and a dose-response relationship was demonstrated by the statistically significant trend test (p < 0.05). Nevertheless, the corresponding IMF (34x 10-6) remained substantially below the GEF of +126 x 10-6, consequently, these results did not meet the criteria of a positive response. Since no similar increase in the MF was noted in the first experiment performed under the same experimental conditions, even up to a higher dose-level of 200 µg/mL, it was considered not to be biologically relevant.
Consequently, the overall results met the criteria of a negative response.
The test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
- 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
- Study period:
- 12 February 2016 - 18 April 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable (not a gene mutation assay)
- Species / strain / cell type:
- other: human lymphocytes
- Details on mammalian cell type (if applicable):
- Cultures of human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours.
Cultures of human lymphocytes were prepared from whole blood sample obtained from one healthy, non-smoking donor and collected into heparinized sterile tube. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route
- Test concentrations with justification for top dose:
- Top dose-levels limited by precipitation of the test item in the culture medium.
The dose-levels selected for the treatment of the first experiment with and without S9 mix were 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL.
The dose-levels selected for the second experiment with and without S9 mix were 3.91, 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL. - Vehicle / solvent:
- - Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: since DMSO allowed solubilization of the test item. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: mitomycin C (-S9 mix); cyclophosphamide (+S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION:
Without S9 mix:
- 3-hour treatment and 20-hour harvest time in the first experiment
- continuous treatments and 20- and 44-hour harvest times in the second experiment
With S9 mix:
- 3-hour treatment and 20-hour harvest time in the first experiment
- 3-hour treatments and 20- and 44-hour harvest times in the second experiment
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF CELLS EVALUATED: 300 metaphases/dose-level
DETERMINATION OF CYTOTOXICITY
- Method: the cytotoxicity of the test item was assessed by the observation of mitotic inhibition in treated cultures in comparison to vehicle control cultures.
The Mitotic Index (MI) was evaluated in each culture, in each experiment, by scoring the number of cells in mitosis on a total of 1000 cells per culture.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes. - Evaluation criteria:
- The biological relevance of the results was always taken into account when evaluating results.
Evaluation of a positive response:
a test item is considered clearly positive for inducing chromosomal aberrations if, in any of the experimental conditions examined:
- a statistically significant increase in the frequency of cells with structural chromosome aberration is observed at one or more dose-levels,
- a dose-response relationship is demonstrated by a statistically significant trend test,
- any of the results are above the historical data range of the corresponding vehicle control.
Evaluation of a negative response:
a test item is clearly considered negative for inducing chromosomal aberrations if, in all experimental conditions, none of the criteria for a positive response are met.
If the criteria of a positive or a negative response are only partially met, results were evaluated on a case by case basis. - Statistics:
- For each condition of the cytogenetic experiment, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the X2 test unless treated culture data were lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance.
To assess the dose-response trend, a linear regression was performed between the frequencies of cells with structural chromosome aberration (excluding gaps) and the dose-levels. - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Experiments without S9 mix: (cf tables 7.6.1/1 to 7.6.1/3 in chapter any other information on results)
Cytotoxicity
No noteworthy decrease in the Mitotic Index was noted at any of the tested dose-levels following the 3-, 20- or 44 hour treatment.
Metaphase analysis
The dose-levels selected for metaphase analysis were as follows:
- 31.3, 62.5 and 125 µg/mL for the 3-hour treatment, the latter being the lowest dose-level showing precipitate in the culture medium,
- 15.6, 31.3 and 62.5 µg/mL for the 20-hour treatment, the latter being the lowest dose-level showing precipitate in the culture medium,
- 62.5 µg/mL for the 44-hour treatment, this dose-level being the lowest one showing precipitate in the culture medium.
No statistically significant or dose-related increases in the frequency of cells with structural chromosomal aberrations were observed in either experiment. All frequencies of cells with structural chromosomal aberrations obtained from the test item-treated cultures remained within the corresponding vehicle controls historical ranges. These results met the criteria of a negative response.
Experiments with S9 mix:(cf tables 7.6.1/4 to 7.6.1/6 in chapter any other information on results)
Cytotoxicity
At the 20-hour harvest time in the first experiment, slight decreases in the Mitotic Index were noted at dose-levels > or = to 250 µg/mL (29 to 34% decrease).
At the 20- and 44-hour harvest times in the second experiment, no noteworthy decrease in the Mitotic Index was noted at any of the tested dose-levels.
Metaphase analysis
The dose-levels selected for metaphase analysis were as follows:
- 31.3, 62.5 and 125 µg/mL for the first experiment, the latter being the lowest dose-level showing precipitate in the culture medium,
- 15.6, 31.3 and 62.5 µg/mL for the 20-hour harvest time in the second experiment, the latter being the lowest dose-level showing precipitate in the culture medium,
- 62.5 µg/mL for the 44-hour harvest time in the second experiment, this dose-level being the lowest one showing precipitate in the culture medium.
In the first experiment (20-hour harvest time), frequencies of cells with structural chromosomal aberrations were sometimes slightly above the corresponding vehicle control historical range (2.7% versus [0-2.5%] for the historical data); these findings were not reproduced in the second experiment performed in the same conditions (20-hour harvest time) (cf tables 7.6.1/7 in chapter any other information on results for historical data).
Since, no statistical differences were observed relative to the vehicle controls and no dose-response relationship was demonstrated in either experiment, the overall results were considered to meet the criteria of a negative response
In the second experiment at the 44-hour harvest time (Table 12), both control and treated cultures showed frequencies of cells with structural chromosomal aberrations slightly above the corresponding vehicle control historical range (2.0 and 2.7% versus [0.0-1.5%] for the historical data).Considering the very narrow range of historical data available for this test condition, ranges of historical data available for other conditions (up to 3.5% for the 20-hour treatment without S9 mix), as well as the lack of any statistical difference between the vehicle control and the test item treated cultures, these results were interpreted as a negative response (cf tables 7.6.1/7 in chapter any other information on results for historical data).
- Conclusions:
- The substance was not considered to be clastogenic to the human lymphocytes in the in vitro chromosomal aberration assay either in the presence or in the absence of a rat liver metabolizing system.
- Executive summary:
The objective of this study was to evaluate the potential of the test item, to induce chromosome aberrations in cultured human lymphocytes. The study was performed according to OECD guideline No. 473 and in compliance with the Principles of Good Laboratory Practice.
The test item, dissolved in dimethylsulfoxide (DMSO), was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of available pH, osmolality and solubility data. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at, for about 48 hours.
In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Then, the cultures were incubated in fresh medium until harvest. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
The second experiment was performed as follows:
- without S9 mix, cells were exposed continuously to the test or control items until harvest,
- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Then, the cultures were incubated in fresh medium at 37°C until harvest.
Harvest times were 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.
Three hours before harvest, each culture was treated with a Colcemid® solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The MI was first of all evaluated on each slide. Then, structural and numerical aberrations were recorded for cultures treated in the absence and presence of S9 mix with three dose-levels of the test item, the vehicle and positive controls.
Based on available solubility data, the dose-level of 1000 µg/mL, obtained using a test item concentration of 100 mg/mL and a treatment volume of 1% (v/v), was expected to produce precipitation in the culture medium. Thus the dose-levels selected for the treatment of the first experiment with and without S9 mix were 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL.
Based on the results of the first experiment, the dose-levels selected for the second experiment with and without S9 mix were 3.91, 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL.
At the highest tested dose-levels of 1000 µg/mL, the pH of the culture medium was approximately 7.7 (as for the vehicle control) and the osmolality was 428 mOsm/kg H2O (454 mOsm/kg for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions.
The frequencies of cells with structural chromosome aberrations for the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered to be valid.
At the end of the treatment periods, a precipitate was observed in the culture medium at dose-levels superior or egal to 125 µg/mL in the first experiment and superior or egal to 62.5 µg/mL in the second experiment.
Experiments without S9 mix
Cytotoxicity
No noteworthy decrease in the MI was noted at any of the tested dose-levels following the 3-, 20- or 44-hour treatment.
Metaphase analysis
The dose-levels selected for metaphase analysis were as follows:
. 31.3, 62.5 and 125 µg/mL for the 3-hour treatment, the latter being the lowest dose-level showing precipitate in the culture medium,
. 15.6, 31.3 and 62.5 µg/mL for the 20-hour treatment, the latter being the lowest dose-level showing precipitate in the culture medium,
. 62.5 µg/mL for the 44-hour treatment, this dose-level being the lowest one showing precipitate in the culture medium.
No statistically significant or dose-related increases in the frequency of cells with structural chromosomal aberrations were observed in either experiment. All frequencies of cells with structural chromosomal aberrations obtained from the test item treated cultures remained within the corresponding vehicle controls historical ranges. These results met the criteria of a negative response.
Experiments with S9 mix
Cytotoxicity
At the 20-hour harvest time in the first experiment, slight decrease in the MI were noted at dose-levels superior or egal to 250 µg/mL (29 to 34% decrease).
At the 20- and 44-hour harvest times in the second experiment, no noteworthy decrease in the MI was noted at any of the tested dose-levels.
Metaphase analysis
The dose-levels selected for metaphase analysis were as follows:
- 31.3, 62.5 and 125 µg/mL for the first experiment, the latter being the lowest dose-level showing precipitate in the culture medium,
- 15.6, 31.3 and 62.5 µg/mL for the 20-hour harvest time in the second experiment, the latter being the lowest dose-level showing precipitate in the culture medium,
- 62.5 µg/mL for the 44-hour harvest time in the second experiment, this dose-level being the lowest one showing precipitate in the culture medium.
In the first experiment (20-hour harvest time), frequencies of cells with structural chromosomal aberrations were sometimes slightly above the corresponding vehicle control historical range (2.7% versus [0-2.5%] for the historical data); these findings were not reproduced in the second experiment performed in the same conditions (20-hour harvest time).
Since, no statistical differences were observed relative to the vehicle controls and no dose-response relationship was demonstrated in either experiment, the overall results were considered to the meet criteria of a negative response.
In the second experiment at the 44-hour harvest time, both control and treated cultures showed frequencies of cells with structural chromosomal aberrations slightly above the corresponding vehicle control historical range (2.0 and 2.7% versus [0.0-1.5%] for the historical data). Considering the very narrow range of historical data available for this test condition, ranges of historical data available for other conditions (up to 3.5% for the 20-hour treatment without S9 mix), as well as the lack of any statistical difference between the vehicle control and the test item treated cultures, these results were interpreted as a negative response.
The test item, did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a rat liver metabolizing system.
Referenceopen allclose all
7.6.1/3: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the first test (direct plate incorporation method)
20231250 Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
E. coli WP2 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
26.7 |
2.5 |
13.3 |
2.9 |
39.0 |
1.0 |
161.7 |
6.7 |
170.3 |
5.7 |
5 |
27.7 |
4.7 |
17.3 |
2.5 |
35.0 |
6.1 |
169.7 |
20.4 |
153.3 |
9.8 |
15 |
30.0 |
10.4 |
19.3 |
2.1 |
34.3 |
2.5 |
159.7 |
19.4 |
167.7 |
13.1 |
50 |
28.0 |
7 |
16.7 |
2.9 |
45.3 |
4.2 |
153.3 |
18.7 |
177.3 |
5.5 |
150 |
22.0 |
3.5 |
15.0 |
1.7 |
42.0 |
4.4 |
152.0 |
11.1 |
169.3 |
7.6 |
500 |
26.0 |
3.6 |
21.0 |
1.0 |
47.3 |
4.7 |
146.0 |
7.9 |
160.3 |
10.1 |
1500 |
25.3 |
2.9 |
13.3 |
4.0 |
34.0 |
2.0 |
161.3 |
22.7 |
178.3 |
11.0 |
5000 |
24.0 |
3.6 |
9.7 |
1.5 |
28.7 |
5.5 |
155.3 |
4.5 |
164.7 |
8.1 |
Positive control |
1063.7 |
79.7 |
913.0 |
138.4 |
256.3 |
114.6 |
961.3 |
79.0 |
2531.0 |
132.7 |
*Solvent control = negative control: 100 µL DMSO
7.6.1/4: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the first test (direct plate incorporation method)
20231250 Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
E. coli WP2 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
27.3 |
0.6 |
28.7 |
3.1 |
54.7 |
6.7 |
197.7 |
11.1 |
196.3 |
3.1 |
5 |
24.0 |
3.5 |
27.3 |
5.8 |
51.7 |
6.1 |
151.3 |
13.0 |
211.3 |
28.5 |
15 |
23.3 |
2.3 |
21.3 |
4.6 |
50.0 |
6.6 |
158.7 |
11.0 |
180.7 |
23.1 |
50 |
25.0 |
2.6 |
30.3 |
5.8 |
50.7 |
5.0 |
166.7 |
12.7 |
196.3 |
4.9 |
150 |
21.7 |
2.1 |
34.0 |
4.6 |
55.0 |
4.0 |
175.7 |
6.5 |
203.0 |
15.7 |
500 |
27.0 |
4.6 |
32.7 |
2.1 |
54.3 |
5.1 |
176.3 |
14.4 |
167.0 |
13.5 |
1500 |
26.7 |
3.1 |
27.3 |
3.8 |
39.7 |
7.8 |
182.0 |
13.1 |
165.3 |
12.1 |
5000 |
22.7 |
2.1 |
25.3 |
1.2 |
46.0 |
2.0 |
156.7 |
10.7 |
159.0 |
10.5 |
Positive control |
307.3 |
23.9 |
193.0 |
12.5 |
206.7 |
17.5 |
3014.7 |
216.5 |
601.3 |
24.8 |
*Solvent control = negative control: 100 µL DMSO
7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the second test (preincubation method)
20231250 Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
E. coli WP2 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
23.0 |
0.0 |
14.0 |
1.7 |
41.0 |
2.6 |
161.0 |
18.7 |
195.7 |
7.5 |
5 |
20.3 |
3.8 |
16.3 |
0.6 |
37.0 |
1.7 |
172.3 |
15.6 |
160.7 |
22.6 |
15 |
15.3 |
3.8 |
16.7 |
1.5 |
38.0 |
2.6 |
150.0 |
12.8 |
182.0 |
21.8 |
50 |
16.0 |
1.7 |
18.0 |
3.5 |
42.0 |
8.0 |
139.3 |
12.2 |
194.7 |
15.3 |
150 |
24.3 |
2.9 |
16.0 |
1.0 |
43.0 |
2.6 |
137.3 |
6.0 |
188.7 |
6.7 |
500 |
22.3 |
4.0 |
13.0 |
1.7 |
39.7 |
9.0 |
121.3 |
3.2 |
173.7 |
2.3 |
1500 |
20.7 |
1.5 |
12.7 |
1.5 |
34.7 |
2.5 |
139.7 |
5.5 |
157.3 |
22.1 |
5000 |
16.7 |
2.1 |
11.3 |
1.5 |
27.0 |
6.9 |
138.7 |
15.0 |
189.0 |
11.5 |
Positive control |
987.0 |
23.4 |
918.3 |
74.7 |
378.3 |
35.1 |
802.0 |
18.0 |
1878.0 |
162.4 |
*Solvent control = negative control: 100 µL DMSO
Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the second test (preincubation method)
20231250 Concentration |
TA 1535 |
TA1537 |
TA 98 |
TA 100 |
E. coli WP2 |
|||||
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
Mean |
Standard deviation |
|
0* |
19.0 |
2.6 |
27.3 |
1.5 |
49.3 |
1.2 |
195.7 |
23.4 |
220.0 |
10.1 |
5 |
14.3 |
3.8 |
29.7 |
2.1 |
52.7 |
5.8 |
193.0 |
23.5 |
221.3 |
36.6 |
15 |
21.7 |
2.1 |
29.0 |
5.0 |
51.0 |
10.8 |
174.3 |
16.8 |
205.3 |
19.4 |
50 |
18.3 |
4.6 |
30.7 |
2.3 |
55.0 |
10.5 |
156.0 |
3.6 |
202.7 |
24.1 |
150 |
17.7 |
5.9 |
24.7 |
2.3 |
54.0 |
1.0 |
171.3 |
5.0 |
190.3 |
39.7 |
500 |
17.7 |
5.5 |
36.7 |
4.7 |
51.3 |
10.0 |
171.0 |
2.6 |
188.3 |
13.2 |
1500 |
17.3 |
3.1 |
24.0 |
2.6 |
44.7 |
2.3 |
149.7 |
9.9 |
170.0 |
15.7 |
5000 |
15.0 |
2.6 |
20.0 |
6.6 |
42.0 |
6.2 |
129.3 |
26.1 |
158.3 |
10.2 |
Positive control |
287.3 |
26.1 |
138.0 |
28.6 |
187.7 |
9.9 |
4437.7 |
129.6 |
713.0 |
55.1 |
*Solvent control = negative control: 100 µL DMSO
7.6.1/1 First experiment without S9 mix : chromosome aberration (3-hour treatment, 20-hour harvest)
Doses µg/mL |
Slide nb |
Nb of cells scored |
NA |
Structural chromosome aberrations (type and number) |
Cells with structural chromosome aberrations |
|
|||||||||||
G |
chromatid |
chromosome |
MA |
PU |
Total +G |
Total -G |
Nb +G |
Frequency (%) +G |
Nb -G |
Frequency (%) -G |
|||||||
D |
Exch |
D |
Exch |
||||||||||||||
0 |
7C1 |
150 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
3 |
3 |
1 |
1.0 |
1 |
1.0 |
|
2C2 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
||||||
31.3 |
1C1 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
6 |
6 |
2 |
2.0 |
2 |
2.0 |
|
19C2 |
150 |
0 |
0 |
2 |
0 |
1 |
0 |
1 |
0 |
4 |
4 |
||||||
62.5 |
37C1 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
4 |
4 |
2 |
1.3 |
2 |
1.3 |
|
25C2 |
150 |
1 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
2 |
2 |
||||||
125 P |
15C1 |
150 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
6 |
5 |
1 |
2.0 |
1 |
1.7 |
|
32C2 |
150 |
0 |
1 |
2 |
0 |
2 |
0 |
0 |
0 |
5 |
4 |
||||||
MMC 2 µg / mL |
34C1 |
50 |
0 |
1 |
25 |
13 |
5 |
0 |
3 |
0 |
90 |
88 |
29 |
56.0 |
28 |
55.0 *** |
|
4C2 |
50 |
0 |
1 |
26 |
12 |
3 |
0 |
1 |
0 |
27 |
27 |
||||||
7.6.1/2 Second experiment without S9 mix : chromosome aberration (20-hour treatment, 20-hour harvest)
Doses µg/mL |
Slide nb |
Nb of cells scored |
NA |
Structural chromosome aberrations (type and number) |
Cells with structural chromosome aberrations
|
|||||||||||
G |
chromatid |
chromosome |
MA |
PU |
Total +G |
Total -G |
Nb +G |
Frequency (%)
+G |
Nb -G |
Frequency (%)
-G |
||||||
D |
Exch |
D |
Exch |
|||||||||||||
0 |
45C1 |
150 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
5 |
5 |
1 |
1.7 |
1 |
1.7 |
59C2 |
150 |
0 |
0 |
4 |
0 |
0 |
0 |
0 |
0 |
4 |
4 |
|||||
15.6 |
53C1 |
150 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
5 |
4 |
1 |
1.3 |
1 |
1.3 |
70C2 |
150 |
0 |
1 |
2 |
0 |
1 |
0 |
0 |
0 |
3 |
3 |
|||||
31.3 |
49C1 |
150 |
0 |
0 |
2 |
0 |
1 |
0 |
0 |
0 |
7 |
7 |
3 |
2.3 |
3 |
2.3 |
76C2 |
150 |
0 |
0 |
3 |
0 |
1 |
0 |
0 |
0 |
4 |
4 |
|||||
62.5 P |
69C1 |
150 |
1 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
4 |
4 |
2 |
1.3 |
2 |
1.3 |
65C2 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
|||||
MMC 0.2 µg / mL |
48C1 |
50 |
0 |
0 |
15 |
4 |
1 |
0 |
0 |
0 |
30 |
30 |
14 |
24.0 |
14 |
24.0 *** |
78C2 |
50 |
0 |
0 |
5 |
3 |
2 |
0 |
0 |
0 |
10 |
10 |
|||||
7.6.1/3 Second experiment without S9 mix : chromosome aberration (44-hour treatment, 44-hour harvest)
Doses µg/mL |
Slide nb |
Nb of cells scored |
NA |
Structural chromosome aberrations (type and number) |
Cells with structural chromosome aberrations
|
|||||||||||
G |
chromatid |
chromosome |
MA |
PU |
Total +G |
Total -G |
Nb +G |
Frequency (%)
+G |
Nb -G |
Frequency (%)
-G |
||||||
D |
Exch |
D |
Exch |
|||||||||||||
0 |
85C1 |
150 |
0 |
1 |
2 |
0 |
0 |
0 |
0 |
0 |
5 |
4 |
3 |
1.7 |
2 |
1.3 |
96C2 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
|||||
62.5 P |
106C1 |
150 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
4 |
4 |
1 |
1.3 |
1 |
1.3 |
7.6.1/4 First experiment with S9 mix : chromosome aberration (3-hour treatment, 20-hour harvest)
Doses µg/mL |
Slide nb |
Nb of cells scored |
NA |
Structural chromosome aberrations (type and number) |
Cells with structural chromosome aberrations
|
|||||||||||
G |
chromatid |
chromosome |
MA |
PU |
Total +G |
Total -G |
Nb +G |
Frequency (%)
+G |
Nb -G |
Frequency (%)
-G |
||||||
D |
Exch |
D |
Exch |
|||||||||||||
0 |
21C1 |
150 |
0 |
0 |
3 |
1 |
0 |
0 |
0 |
0 |
8 |
8 |
4 |
2.7 |
4 |
2.7 |
13C2 |
150 |
0 |
0 |
4 |
0 |
0 |
0 |
0 |
0 |
4 |
4 |
|||||
31.3 |
36C1 |
150 |
0 |
0 |
5 |
0 |
1 |
0 |
0 |
0 |
9 |
9 |
5 |
2.7 |
5 |
2.7 |
20C2 |
150 |
0 |
0 |
3 |
0 |
0 |
0 |
0 |
0 |
3 |
3 |
|||||
62.5 |
28C1 |
150 |
1 |
0 |
6 |
0 |
0 |
0 |
0 |
0 |
10 |
8 |
6 |
3.0 |
6 |
2.7 |
16C2 |
150 |
1 |
2 |
2 |
0 |
0 |
0 |
0 |
0 |
3 |
2 |
|||||
125 P |
3C1 |
150 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
4 |
4 |
1 |
1.3 |
1 |
1.3 |
39C2 |
150 |
0 |
0 |
1 |
0 |
2 |
0 |
0 |
0 |
3 |
3 |
|||||
CPA 12.5 µg / mL |
31C1 |
50 |
0 |
0 |
15 |
2 |
1 |
0 |
0 |
0 |
40 |
40 |
15 |
32.0 |
15 |
32.0 *** |
23C2 |
50 |
1 |
0 |
16 |
4 |
1 |
0 |
1 |
0 |
17 |
17 |
|||||
7.6.1/5 Second experiment with S9 mix : chromosome aberration (3-hour treatment, 20-hour harvest)
Doses µg/mL |
Slide nb |
Nb of cells scored |
NA |
Structural chromosome aberrations (type and number) |
Cells with structural chromosome aberrations
|
|||||||||||
G |
chromatid |
chromosome |
MA |
PU |
Total +G |
Total -G |
Nb +G |
Frequency (%)
+G |
Nb -G |
Frequency (%)
-G |
||||||
D |
Exch |
D |
Exch |
|||||||||||||
0 |
51C1 |
150 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
3 |
3 |
1 |
1.0 |
1 |
1.0 |
67C2 |
150 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
2 |
2 |
|||||
15.6 |
80C1 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
5 |
5 |
2 |
1.7 |
2 |
1.7 |
55C2 |
150 |
0 |
0 |
3 |
0 |
0 |
0 |
0 |
0 |
3 |
3 |
|||||
31.3 |
50C1 |
150 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
3 |
3 |
2 |
1.0 |
2 |
1.0 |
72C2 |
150 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
|||||
62.5 P |
79C1 |
150 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
3 |
3 |
1 |
1.0 |
1 |
1.0 |
81C2 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
|||||
CPA 12.5 µg / mL |
47C1 |
50 |
0 |
1 |
13 |
1 |
3 |
0 |
0 |
0 |
37 |
34 |
15 |
28.0 |
14 |
26 *** |
68C2 |
50 |
0 |
2 |
10 |
3 |
4 |
0 |
0 |
0 |
13 |
12 |
|||||
7.6.1/6 Second experiment with S9 mix : chromosome aberration (3-hour treatment, 44-hour harvest)
Doses µg/mL |
Slide nb |
Nb of cells scored |
NA |
Structural chromosome aberrations (type and number) |
Cells with structural chromosome aberrations
|
|||||||||||
G |
chromatid |
chromosome |
MA |
PU |
Total +G |
Total -G |
Nb +G |
Frequency (%)
+G |
Nb -G |
Frequency (%)
-G |
||||||
D |
Exch |
D |
Exch |
|||||||||||||
0 |
92C1 |
150 |
0 |
0 |
3 |
0 |
1 |
0 |
0 |
0 |
6 |
6 |
4 |
2.0 |
4 |
2.0 |
89C2 |
150 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
|||||
62.5 P |
93C1 |
150 |
0 |
0 |
2 |
0 |
1 |
0 |
0 |
0 |
8 |
8 |
3 |
2.7 |
3 |
2.7 |
105C2 |
150 |
0 |
0 |
5 |
0 |
0 |
0 |
0 |
0 |
5 |
5 |
|||||
Nb: number, NA: Numerical Aberrations, G: Gap, D: Deletion, Exch: Exchange, MA: Multiple Aberrations, PU: Pulverization
C1: Culture 1 (Female) 0: vehicle control (DMSO)
C2: culture 2 (Female) MMC: Mitomycin C CPA: Cyclophosphamide
P: Precipitate observed in the culture medium at the end of treatment
Statistical analysis : chi-2 test ***: p<0.001(performed only for cells with structural aberrations excluding gaps)
7.6.1/7 Historical data In vitro mammalian chromosome aberration test in cultured human lymphocytes (from 1 june 2012 to 28 august 2015)
Experiments without S9
|
Vehicle control |
MMC |
||||||||
Treatment duration |
Short treatment |
Continuous treatment |
Short treatment |
Continuous treatment |
||||||
Harvest time |
20 hours |
20 hours |
44 hours |
20 hours |
20 hours |
|||||
Parameter |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
n |
30 |
30 |
31 |
30 |
14 |
14 |
30 |
17 |
31 |
20 |
Mean |
11.1 |
0.7 |
8.5 |
1.1 |
8.5 |
1.0 |
3.0 |
38.9 |
5.3 |
20.7 |
SD |
3.2 |
0.6 |
2.8 |
0.9 |
2.2 |
0.7 |
1.6 |
9.1 |
2.6 |
6.1 |
Lower CL 95% |
9.9 |
0.4 |
7.5 |
0.7 |
7.2 |
0.6 |
2.4 |
34.3 |
4.4 |
17.9 |
Upper CL 95% |
12.3 |
0.9 |
9.5 |
1.4 |
9.8 |
1.5 |
3.6 |
43.6 |
6.3 |
23.6 |
5th Percentile |
5.1 |
0.0 |
3.3 |
0.0 |
6.0 |
0.0 |
1.2 |
20.0 |
2.4 |
9.4 |
Median |
11.0 |
0.5 |
7.8 |
1.0 |
8.0 |
1.0 |
2.6 |
39.0 |
5.1 |
20.8 |
95% Percentile |
15.3 |
2.0 |
13.0 |
3.0 |
13.1 |
2.0 |
5.9 |
60.0 |
11.4 |
31.0 |
Min |
4.8 |
0.0 |
3.0 |
0.0 |
6.0 |
0.0 |
0.9 |
20.0 |
2.4 |
5.5 |
Max |
19.0 |
2.0 |
13.2 |
3.5 |
13.1 |
2.0 |
6.6 |
60.0 |
11.8 |
33.0 |
7.6.1/7 Historical data In vitro mammalian chromosome aberration test in cultured human lymphocytes (from 1 june 2012 to 28 august 2015)
Experiments withS9
|
Vehicle control |
CPA 12.5 µg/mL |
CPA 25 µg/mL |
|||||
Treatment duration |
Short treatment |
Short treatment |
Short treatment |
Short treatment |
||||
Harvest time |
20 hours |
44 hours |
20 hours |
20 hours |
||||
Parameter |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
Mitotic index |
% ab. cells |
n |
48 |
48 |
12 |
12 |
48 |
36 |
46 |
12 |
Mean |
9.6 |
0.8 |
8.3 |
0.5 |
5.5 |
23.1 |
3.4 |
24.7 |
SD |
3.0 |
0.7 |
2.4 |
0.5 |
2.0 |
7.3 |
1.4 |
7.0 |
Lower CL 95% |
8.7 |
0.6 |
6.8 |
0.2 |
4.9 |
20.6 |
3.0 |
20.2 |
Upper CL 95% |
10.4 |
1.0 |
9.8 |
0.9 |
6.0 |
25.6 |
3.8 |
29.2 |
5th Percentile |
5.6 |
0.0 |
4.4 |
0.0 |
2.1 |
13.0 |
1.8 |
15.5 |
Median |
9.6 |
0.5 |
8.9 |
0.5 |
5.4 |
22.5 |
3.3 |
24.5 |
95% Percentile |
14.6 |
2.0 |
11.1 |
1.5 |
8.4 |
39.0 |
6.0 |
41.0 |
Min |
2.9 |
0.0 |
4.4 |
0.0 |
1.4 |
8.5 |
1.4 |
15.5 |
Max |
14.8 |
2.5 |
11.1 |
1.5 |
12.8 |
40.0 |
8.2 |
41.0 |
% ab.cells: frequency of aberrant cells excluding cells containing only gaps
CL: Confidence Limit CPA: Cyclophosphamide Max: Maximal value
Min: Minimal value MMC: Mitomycin C at 3.0 µg/mL for the short treatment or 0.3µg/mL for the continuous treatment
n: number of values (the values used are the mean of duplicate cultures), SD: Standard Deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in bacteria
The potential of the substance to induce reverse mutation in bacteria was assessed using Salmonella typhimurium TA1535, TA 1537, TA 100 ,TA 98 and E. coli WP2 according to the OECD guideline 471.The study was conducted in compliance with Good Laboratory Practices.
A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system.
The both methods of direct plate incorporation or with a preincubation step were tested during the two experiments. After 48 -72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
The highest concentration chosen was 5000 µg/plate as it was the standard limit concentration recommended in the international guidelines at this time. The selected treatment-levels were 5, 15, 50,150, 500, 1500 and 5000 µg/plate in both mutagenicity experiments.
Precipitate was observed in the petri plates at dose-levels equal or higher than 1500 µg/plate for all strains in both experiments .No noteworthy toxicity was noted at any dose-levels either with or without S9 mix. In the two independent assays, no significant increase in the mean number of revertants was noted in the bacterial strains tested in the presence of the test substance neither with nor without metabolic activation.
it was concluded that the substance did not show any mutagenic activity in the bacterial reverse mutation test.
Gene mutation in mammalian cells
An in vitro mouse lymphoma assay was conducted to evaluate forward mutation induction potential of the test substance at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The study was conducted according to the OECD Guideline 490 and in compliance with the principles of Good Laboratory Practices. After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system .
Cultures of 20 mL at 5 x 105cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. The test item was dissolved in dimethylsulfoxide.
Since the test item was cytotoxic but also poorly soluble in the culture medium, the highest dose-level for the main test was selected on the basis of the level of precipitate.
The dose-levels were 6.25, 12.5, 25, 50,100 and 200 µg/mL for both mutagenicity experiments without S9 mix. A precipitate was observed in the culture medium at dose-levels of 50 µg/mL or greater at the end of the 3-hour treatment and at 25 µg/mL or greater at the end of the 24-hour treatment. Following the 3-hour and 24 -hour treatments, no cytotoxicity was induced at any of the tested dose-levels. No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control, following either the 3- or the 24-hour treatment.
The dose-levels were 6.25, 12.5, 25, 50, 100 and 200 µg/mL for the first mutagenicity experiments with S9 mix and 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for the second one. A precipitate was observed in the culture medium at dose-levels of 50 µg/mL or greater but no toxicity was noted as shown by the absence of any noteworthy decrease in the Adjusted Relative Total Growth.
No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control, in the first experiment. In the second experiment, a very slight increase in the mutation frequancy (MF) was observed at the dose-level of 100 µg/mL and a dose-response relationship was demonstrated by the statistically significant trend test (p < 0.05). Nevertheless, the corresponding IMF (34x 10-6) remained substantially below the GEF of +126 x 10-6, consequently, these results did not meet the criteria of a positive response. Since no similar increase in the MF was noted in the first experiment performed under the same experimental conditions, even up to a higher dose-level of 200 µg/mL, it was considered not to be biologically relevant. Consequently, the overall results met the criteria of a negative response.
Chromosomal aberrations in mammalian cells
An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to OECD Guideline 473 in compliance with Good Laboratory Practices.
Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. The highest dose-level in the first experiment was selected on the basis of pH, osmolality and solubility data. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of the mitotic index (MI) in the first experiment was also taken into account. The following concentrations were therefore selected for the two independent tests evaluating the metaphase analysis:
- Test 1(3-h treatment with and without S9 mix) at dose levels of 0, 31.3, 62.5 and 125 µg/mL,
- Test 2( 3-h treatment with S9 mix and 20 -hr treatment without S9 mix) at dose levels of 0, 15.6, 31.3 and 62.5 µg/mL and (44 -h treatment with and without S9 mix) at 0 and 62.5 µg/mL .
Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests without S9 mix, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations when compared with the vehicle control. In the first experiment with S9 mix, frequencies of cell with structural chromosomal aberrations were sometimes slightly above the corresponding vehicle control historical range (2.7% versus [0 -2.5%] for the historical data), these findings were not reproduced in the second experiment performed in the same conditions. Since no statistical differences were observed relative to the vehicle controls and no dose-response relationship was demonstrated in either experiment, the overall results were considered to meet the criteria of a negative response.
In the 44 -hour treatment experiment with S9 mix, both control and treated cultures showed frequencies of cells with structural chromosomal aberrations slightly above the corresponding vehicle control historical range (2.0 and 2.7% versus [0.0 -1.5%] for the historical data). Considering the very narrow range of historical data available for this test condition, ranges of historical data available for other conditions (up to 3.5% for the 20-hour treatment without S9 mix) as well as the lack of any statistical difference between the vehicle control and the test item-treated cultures, these results were assessed as a negative response.
All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay.
Justification for classification or non-classification
Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicity according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
