Registration Dossier
Registration Dossier
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EC number: 475-900-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Reverse Mutation Assay (Ames test): The results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicated that ETFBO was not mutagenic under the conditions employed in this study.
In Vitro Mammalian Chromosomal Aberration Test: Under the conditions used in both independent chromosome aberration tests, the test substance ETFBO was not clastogenic for Chinese Hamster Ovary (CHO) cells.
In Vitro Mammalian Cell Micronucleus Test: Under the conditions used in both independent in vitro micronucleus tests, the test substance ETFBO was neither clastogenic nor aneugenic to cultured human lymphocytes.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 9 Aug.-7 Sept. 2005
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The study was conducted according to an internationally recognised method, and under GLP. However, the test item identification is incomplete (unspecified cis-trans isomery, therefore identified under the generic CAS 17129-06-5). Therefore validation applies with restrictions.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Remarks:
- The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certificate from 2004-08-19
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium strains: histidine requirement for growth
E. coli strains: tryptophan requirement for growth - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- - Source of S9: The batch of S9 was prepared according to Ames et al. (1975) and Maron and Ames (1983) as follows. Male Wistar rats (n =12; obtained from Charles River Deutschland , Sulzfeld, Germany) were injected intraperitoneally with a single dose of Aroclor 1254 (nominal dose of 500 mg/kg body weight) in soy bean oil (20% w/v). The rats were provided with tap water and the Institute's stock diet ad libitum. Five days after the injection of Aroclor 1254 the rats were killed by CO2 asphyxiation. The livers were removed aseptically and immediately put into a cold, sterile 0.15 M KCI solution. After washing in the KCI solution, the livers were weighed, cut into pieces and homogenized in 3 volumes of 0.15 M KCl solution in a Potter-Elvehjem apparatus with a Teflon pestle. The homogenate was centrifuged for 10 minutes at 9,000 g. The supematant, which is called S9, was collected and divided into small aliquots in sterile polypropylene vials. The vials were quickly frozen on dry ice and subsequently stored in a freezer at <-60 °C.
- Method of preparation of S9 mix : On the day of use, aliquots of S9 liver homogenate were thawed and mixed with a NADPH generating system. The S9-mix was kept on ice until use.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCI 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4), NaCI 46 mM, and S9 10 %. 0.5 mL of this S9-mix was used in the experiments with metabolic activation
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was checked for sterility. The protein and cytochrome P-450 content of the S9 fraction were determined according to the method published by Rutten et al. (1987). The protein content of the batch was 21.2 g/L. The cytochrome P450 content of the batch was 19.4 µmol/L. The batch contained 0.91 µmol cytochrome P450 per gram protein. The sterility check of the batch resulted in 0 colonies per 100 µL S9. The batch of S9 met all of the in-house quality criteria. - Test concentrations with justification for top dose:
- According to OECD 471 guideline, cytoxicity and solubility are criteria to be taken into consideration when determining the highest test substance concentration. In this study, a preliminary test to assess the toxicity of the test substance was not performed; therefore, the toxicity investigation was incorporated in the first mutagenicity test.
First test: Five concentrations were tested, ranging from 62 to 5000 µg/plate. The highest concentration of 5 mg/plate was the guideline recommended maximum concentration for soluble non-cytotoxic substances. Highly toxic effects were observed in this first test. According to the guideline recommendation, substances that are cytotoxic already below 5 mg/plate should be tested up to a cytotoxic concentration. A second test was therefore performed at lowest concentrations, still including cytotoxic effects.
Second test: Six concentrations were tested, ranging from 16 to 500 µg/plate. - Vehicle / solvent:
- - Vehicle used: DMSO.
- Justification for choice of vehicle: The absence of mutagenic effects of DMSO was demonstrated through assays performed in the testing laboratory (historical negative control data appended to the report covered assays conducted between May 2002 - March 2005). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- ethylnitrosurea
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate.
- Number of independent experiments : 2 (see above in the field "Test concentrations with justification for top dose").
METHOD OF TREATMENT/ EXPOSURE:
- Test substance formulation: The plate-incorporation method was applied. DMSO was used as a vehicle. Just before use, the test substance was diluted at 50 mg/mL in the first test and 5 mg/mL in the second test (taking the purity of the test substance into account). A clear, light-yellow dilution was obtained in the first test and a clear, colourless dilution was obtained in the second test. In the first test, serial 3-fold dilutions in DMSO were prepared. Five concentrations were tested, ranging from 62 to 5000 µg/plate. In the second test serial 2-fold dilutions in DMSO were prepared. Six concentrations were tested, ranging from 16 to 500 µg/plate. The actual concentrations of the test substance in the test solutions were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
- Exposure conditions: To 2 mL molten top agar (containing 0.6 % agar, 0.5 % NaCI and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophane for the E. coli WP2 uvrA strain), maintained at ca. 46 °C, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the test substance or of the negative or the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). The plates were incubated at ca. 37 °C for approximately 72 hours.
METHODS FOR MEASUREMENT OF CYTOTOXICITY :
Cytotoxicity is defined as a reduction (at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
METHODS FOR MEASUREMENTS OF GENOTOXICIY :
The revertant bacteria his+ and trp+ were counted.
his+ = revertant S. typhimurium able to grow in the absence of histidine.
trp+ = revertant E. coli able to grow in the absence of tryptophan. - Evaluation criteria:
- A test substance is considered to be positive if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates. A test substance is considered to be negative if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points. Both numerical significance and biological relevance are considered together in the evaluation.
- Statistics:
- No statistical analysis was performed .
- Key result
- Species / strain:
- other: all strains (S. typhimurium + E. coli)
- Remarks:
- first test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 185 µg/plate: pinpoint colonies, (slightly) less dense background lawn of the bacterial growth and decrease in the mean number of revertants.
- Vehicle controls validity:
- valid
- Remarks:
- revertant colonies per plate within the acceptability ranges for negative control data.
- Untreated negative controls validity:
- other: not tested
- True negative controls validity:
- other: not tested
- Positive controls validity:
- valid
- Remarks:
- minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.
- Key result
- Species / strain:
- other: all strains (S. typhimurium + E. coli)
- Remarks:
- second test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at and above 250 µg/plate: pinpoint colonies, (slightly) less dense background lawn of the bacterial growth and decrease in the mean number of revertants.
- Vehicle controls validity:
- valid
- Remarks:
- revertant colonies per plate within the acceptability ranges for negative control data.
- Untreated negative controls validity:
- other: not tested
- True negative controls validity:
- other: not tested
- Positive controls validity:
- valid
- Remarks:
- minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.
- Key result
- Species / strain:
- other: S. typhimurium TA 1537 and E. coli WP2 uvrA
- Remarks:
- second test
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 125 µg/plate: slightly less dense background lawn of the bacterial growth.
- Vehicle controls validity:
- valid
- Remarks:
- revertant colonies per plate within the acceptability ranges for negative control data.
- Untreated negative controls validity:
- other: not tested
- True negative controls validity:
- other: not tested
- Positive controls validity:
- valid
- Remarks:
- minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- second test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 125 µg/plate: slightly less dense background lawn of the bacterial growth.
- Vehicle controls validity:
- valid
- Remarks:
- revertant colonies per plate within the acceptability ranges for negative control data.
- Untreated negative controls validity:
- other: not tested
- True negative controls validity:
- other: not tested
- Positive controls validity:
- valid
- Remarks:
- minimum mutation ratio (= number of induced revertants/number of control revertants) within the acceptability ranges for positive control data.
- Additional information on results:
- STUDY RESULTS:
- Signs of toxicity:
* First test: ETFBO was toxic to all strains, in both the absence and presence of S9-mix, at and above 185 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control.
* Second test: ETFBO was slightly toxic to TA 1537 and WP2 uvrA, in the absence of S9-mix, and to TA 98, in both the absence and presence of S9-mix, at 125 µg/plate as was evidenced by a slightly less dense background lawn of the bacterial growth compared to the negative control. Additionally, the test substance ETFBO, was toxic to all strains, in both the absence and presence, at and above 250 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control.
- Signs of genotoxicity (first and second tests): In both the absence and presence of S9-mix in all strains, ETFBO did not cause a dose related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. Except in the second test, in strain TA 1537, in the absence of S9-mix, ETFBO caused a 2.3-fold increase in the mean number of revertant colonies compared to the negative control, at the lowest concentration (16 µg/plate). However, the negative control is relatively low compared to the historical data and since a more than 2-fold increase was observed at only the lowest concentration of the test substance, this was considered to be not biological relevant.
HISTORICAL POSITIVE AND NEGATIVE CONTROL DATA
See in the field below "any other information on results incl. tables". - Conclusions:
- The results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicated that ETFBO was not mutagenic under the conditions employed in this study.
- Executive summary:
The mutagenic activity was investigated in a GPL-compliant study performed according to OECD test guideline 471 (Bacterial Reverse Mutation Assay, Ames test).
The histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophanrequiring Escherichia coli strain WP2uvrA were tested in both the absence and presence of a Iiver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance, ETFBO was diluted in dimethylsulphoxide (DMSO). Two independent tests were performed with all strains, in the absence and the presence of S9-mix, with the following concentrations of the test substance:
- First test: five concentrations ranging from 62 to 5000 µg/plate.
- Second test: six concentrations, ranging from 16 to 500 µg/plate.
Negative controls (DMSO) and positive controls were run simultaneously. All determinations were made in triplicate. The plates were incubated at ca. 37 °C for approximately 72 hours. Subsequently, the his+ and trp+ revertants were counted. A test substance is considered to be positive and thus to induce point mutations by base substitutions or frameshifts in the genome of either S. typhimurium and/or E. coli if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates. A test substance is considered to be negative if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points. Cytotoxicity was also investigated and defined as a reduction (at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
In the first test, ETFBO was toxic to all strains, in both the absence and presence of S9-mix, at and above 185 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control. In the second test, ETFBO was slightly toxic to TA 1537 and WP2 uvrA, in the absence of S9-mix, and to TA 98, in both the absence and presence of S9-mix, at 125 µg/plate as was evidenced by a slightly less dense background lawn of the bacterial growth compared to the negative control. Additionally, the test substance ETFBO, was toxic to all strains, in both the absence and presence, at and above 250 µg/plate, as was evidenced by pinpoint colonies, a (slightly) less dense background lawn of the bacterial growth and a decrease in the mean number of revertants compared to the negative control. In both tests, in the absence and presence of S9-mix in all strains, ETFBO did not cause a dose related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. Except in the second test, in strain TA 1537, in the absence of S9-mix, ETFBO caused a 2.3-fold increase in the mean number of revertant colonies compared to the negative control, at the lowest concentration (16 µg/plate). However, the negative control is relatively low compared to the historical data and since a more than 2-fold increase was observed at only the lowest concentration of the test substance, this was considered to be not biological relevant. The mean number of his+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies; the study was thus considered valid.
The results obtained in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the E. coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicated that ETFBO was not mutagenic under the conditions employed in this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 02 Aug.-14 Sept. 2005
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The study was conducted according to an internationally recognised method, and under GLP. However, the test item identification is incomplete (unspecified cis-trans isomery, therefore identified under the generic CAS 17129-06-5). Therefore validation applies with restrictions.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Remarks:
- The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certificate from 2004-08-19
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: The CHO cells (CHO K-1 line) were obtained from Prof. Dr. A.T. Natarajan, University of Leiden, The Netherlands. This cell line derived from the CHO cells originally isolated from an explant of the ovary of the Chinese hamster (Cricetulus griseus, 2n = 22) by Kac and Puck (1968).
- Normal cell cycle time: The cell-cycle time is 12-14 h.
- Absence of Mycoplasma contamination: Each passage CHO cells in the liquid nitrogen was checked for mycoplasma contamination which was absent.
- Number of passages: 16.
- Methods for maintenance in cell culture: The cells are stored as frozen stock cultures in liquid nitrogen.
- Modal number of chromosomes: The modal chromosome number of these cells is 20-22 (stable aneuploid karyotype).
- Periodically checked for karyotype stability: Each passage CHO cells in the liquid nitrogen was checked for karyotype stability which was stable.
MEDIA USED
The medium for culturing the CHO cells consisted of: Ham's F-12 medium (with Glutamax-I), supplemented with heat-inactivated (45 min, 56 °C) foetal calf serum (10 %), penicillin (100 IU/mL medium) and streptomycin (100 µg/mL medium). - Metabolic activation:
- with and without
- Metabolic activation system:
- - Source of S9
: The S9, used in this study, was part of a batch prepared according to Ames et al. (1975) and Maron and Ames (1983). Male Wistar rats, obtained from Charles River Deutschland, Sulzfeld, Germany, were injected intraperitoneally with a single dose of Aroclor 1254 (nominal dose of 500 mg/kg body weight) in soya bean oil (20% w/v). The rats were provided with tap water and the Institute's stock diet ad libitum. Five days after the inject ion of Aroclor 1254 the rats were killed by CO2 asphyxiation. The livers were removed aseptically and immediately put into a cold, sterile 0.15 M KCI solution. After washing in the KCI solution, the livers were weighed, cut into pieces and homogenized in 3 volumes of 0.15 M KCI solution in a Potter-Elvehjem apparatus with a Teflon pestle. The homogenate was centrifuged for 10 minutes at 9,000 g. The supernatant, which is called S9, was collected and divided into small aliquots in sterile polypropylene vials. The vials were quickly frozen on dry ice and subsequently stored in a freezer at <-60°C.
- Method of preparation of S9 mix: Immediately before use, a S9-mix was prepared by mixing the thawed S9 with a NADPH-generating system.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4) and S9 40 % (v/v). For the first chromosomal aberration test, S9 was used at a concentration of 40 % (v/v). For the second chromosomal aberration test, S9 was used at a concentration of 60 % (v/v). The final concentrations in the culture medium were 4 % (v/v) and 6 % (v/v), respectively for the first and second chromosomal aberration test.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was checked for sterility. The protein and cytochrome P-450 contents of the S9 fraction were determined according to the method published by Rutten et al. The protein content of the batch of S9 was 33 g/L. The cytochrome P-450 content of the batch of S9 was 27.9 µmol/L. The batch of S9 contained 0.85 µmol cytochrome P-450 per gram protein. The sterility check of the batch of S9 resulted in 2 colonies per 100 µL S9. The batch of S9 met all of the in-house quality criteria. - Test concentrations with justification for top dose:
- First test:
1520, 760, 380, 190, 95, 47.5, 23.8, 11.9, 5.9 and 3.0 µg/mL.
The highest concentration of 1520 µg/mL corresponded to 10 mM which was the highest test concentration recommended in OECD 473 guideline for substances triggering no precipitate or limiting cytotoxicity. The conversion from 10 mM to 1520 µg/mL was calculated considering a molecular weight of 152 g/mol. It was however corrected in the study report dealing with the Ames test that the molecular weight of the test substance was 168 g/mol, instead of 152 g/mol. This did not influence the validity of the study because a second chromosome aberration test was performed afterwards at lower concentrations (see below) due to the fact that flocculation (at 1520 and 760 µg/mL with and without S9) and high cytotoxicity (all cells dead or were heavily affected at 1520, 760, 380 and 190 µg/mL with and without S9) were observed at the higher concentrations in the first test.
Second test:
100, 80, 60, 50, 40, 30, 20, 10, 7.5, 5.0, 2.5 and 1.25 µg/mL.
The concentrations for the second test were chosen after the results of the first test. - Vehicle / solvent:
- - Vehicle used: DMSO.
- Justification for choice of vehicle: The absence of chromosome aberration effects of DMSO was demonstrated through assays performed in the testing laboratory (historical negative control data appended to the report covered studies conducted from 2000 to 2003).
- Justification for percentage of vehicle in the final culture medium: The final DMSO concentration in the culture medium was 1% which was in accordance with the guideline recommendation indicating that organic solvents should not exceed 1% (v/v) in the final treatment medium. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate.
- Number of independent experiments : 2 (see above in the field "Test concentrations with justification for top dose").
METHOD OF TREATMENT/ EXPOSURE AND HARVEST:
- Test substance formulation:
* First test: Serial stock concentrations of 152, 76, 38, 19, 9.5, 4.75, 2.38, 1.19, 0.59 and 0.3 mg/mL were freshly prepared in DMSO. The stock concentrations ranging from 152 to 9.5 mg/mL appeared as yellowish clear solutions. 50 µL of each stock concentration were added to the cells. This resulted in final concentrations in the culture medium of 1520, 760, 380, 190, 95, 47.5, 23.8, 11.9, 5.9 and 3.0 µg/mL.
* Second test: Serial stock concentrations of 10, 8, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25 and 0.125 mg/mL were freshly prepared in DMSO. The stock concentrations ranging from 10 to 3 mg/ml, appeared as yellowish clear solutions. 50 µL of each stock concentration were added to the cells. This resulted in final concentrations in the culture medium of 100, 80, 60, 50, 40, 30, 20, 10, 7.5, 5.0, 2.5 and 1.25 µg/mL.
- Preincubation period: Exponentially growing cells were seeded in sterile, screw-capped tissue culture flasks (surface area 25 cm2; 120000 cells per flask) containing 5 mL culture medium and then incubated at ca. 37 °C in humidified air containing 5 % CO2. On the next day (ca. 24 hours after seeding), the exposure started.
- Exposure conditions and duration:
* First test: 50 µL of the vehicle control, 50 µL of each of the dilutions of the test substance or 50 µL of the positive control substance were added to the tissue culture medium in the flasks and the culture medium was checked visually. In the absence of the S9-mix, the culture medium was foetal calf serum. In the presence of the S9-mix, the culture medium with foetal calf serum was replaced by culture medium with penicilline and streptomycine but without foetal calf serum. In addition, in the presence of the S9-mix, 0.5 mL of S9-mix was added to all cultures. The total volume in the flasks was 5 mL. The cultures were incubated at ca. 37 °C in humidified air containing ca. 5 % CO2 and treated for 4 hours (pulse treatment). After 4 hours, the cells and culture medium were checked again. The medium with the test substance was removed, the cells were washed twice with phosphate-buffered saline (pH 7.4) and supplied with 5 mL freshly prepared culture medium. Thereafter, the cells were incubated for an additional 14 hours at ca. 37 °C in humidified air containing ca. 5 % CO2. Two hours before the end of the culture period (18 hours), the cells and culture medium were checked visually.
* Second test: The second (independent) chromosomal aberration test was carried out essentially as described for the first test. ln the presence of S9-mix, the cells were pulse-treated for 4 hours. ln the absence of S9-mix, the cells were treated continuously for 18 hours. The cells of both treatment groups were harvested 18 hours after onset of the treatment.
- Spindle inhibitor and harvest time: Two hours before the end of the total incubation period, the cells were arrested in the metaphase stage of mitosis by the addition of colcemid (final concentration : 0.1 µg/mL). At the end of the total incubation period (18 hours), the cells were harvested by trypsinization, treated for 15 min at ca. 37 °C with a hypotonic solution (1% sodium citrate).
- Methods of slide preparation and staining technique used including the stain used: The cells were fixed with a 3:1 mixture of methanol:glacial acetic acid (two refreshments of the fixative), and transferred to clean microscope slides. Two slides were prepared from each culture. The slides were stained in a 2 % solution of Giemsa, rinsed in water, air-dried and embedded. The slides were coded by a qualified person not involved in scoring the slides, to enable "blind" scoring.
METHODS FOR MEASUREMENT OF CYTOTOXICITY:
- Method: Mitotic Index (MI). At least 1000 nuclei in each culture were examined (500 on each slide) to determine the mitotic index (percentage of cells in mitosis). After the results of the MI scoring and the observations with respect to the quality of the metaphases had been obtained, a selection of the concentrations of the test substance, to be analysed for chromosomal aberrations, was carried out.
METHODS FOR MEASUREMENTS OF GENOTOXICIY:
- Number of cells spread and analysed per concentration: In all treatment groups, in both the first and second (independent) chromosomal aberration tests, three concentrations of the test substance together with the negative and positive controls were selected for the analysis of chromosomal aberrations:
* First test: The concentrations of 5.9, 11.9 and 23.8 µg/mL were analysed for the treatment group with S9-mix and of 11.9, 23.8 and 47.5 µg/mL for the treatment group without S9-mix.
* Second test: The concentrations of 10, 20 and 30 µg/mL were analysed for the treatment group with S9-mix and of 30, 50 and 60 µg/mL for the treatment group without S9-mix.
If possible, the highest concentration should reduce the mitotic index with at least 50 % (but not more than 70 %),when compared to the negative control value or exhibit some other clear indication of cytotoxicity. Subsequently, the cultures of the selected concentrations of the test substance, together with the negative and positive control cultures, were analysed for the induction of structural chromosomal aberrations. For each treatment group, 200 wellspread metaphases per concentration (100 metaphases per culture), each containing 20-22 centromeres, were analysed.
- Criteria for scoring chromosome aberrations and other anomalies: Microscopic examination was used to analyse chromatid-type aberrations (gaps, breaks, fragments, interchanges), chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics) and other anomalies, such as interstitial deletions, endoreduplication, polyploidy and multiple aberrations (> 10 aberrations per cell, excluding gaps), according to the criteria recommended by Savage (1975). - Evaluation criteria:
- A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cells with structural chromosomal aberrations is observed. A test substance is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberrations over the concurrent control frequencies is observed, or if a single positive test point is observed in both tests. A response is considered to be equivocal if the percentage of cells with structural chromosomal aberrations is statistically marginal higher than that of the negative control (0.05 < p < 0.1). A test substance is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points. Both statistical significance and biological relevance are considered together in the evaluation of the results.
N.B. Cells with only gaps (achromatic lesions), heavily damaged cells (cells with multiple aberrations) and cells with polyploidy and endoreduplication were recorded separately and not included in the final assessment of clastogenic activity. - Statistics:
- Data were analysed statistically by Fisher's exact probability test (two-sided) to determine significant differences between treated and control cultures.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- First and second tests
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- For further details, see in the field "Additional information on results".
- Vehicle controls validity:
- valid
- Remarks:
- The numbers of aberrant cells, found in the negative control (DMSO) cultures, were within the historical range.
- Positive controls validity:
- valid
- Remarks:
- The positive control substances, mitomycin C (without S9-mix) and cyclophosphamide (with S9-mix), induced the expected statistically significant increases in the incidence of structural chromosomal aberrations.
- Additional information on results:
- STUDY RESULTS
:
- Cytotoxicity:
* First test: In the pulse treatment group with metabolic activation (S9-mix), the mitotic indices of the concentrations analysed (5.9, 11.9 and 23.8 µg/mL) were reduced to 86%, 73% and 46%, respectively of that of the concurrent negative (DMSO) control. At higher concentrations (47.5 to 1520 µg/mL), the test substance became too toxic for the cells. In the pulse treatment group without metabolic activation (S9-mix), the mitotic indices of all the concentrations analysed (11.9, 23.8 and 47.5 µg/mL) were not reduced when compared to the concurrent negative (DMSO) control. At higher concentrations (95 to 1520 µg/mL), the test substance became too toxic for the cells.
* Second test: In the pulse treatment group with metabolic activation (S9-mix), the mitotic indices of the concentrations analysed (10, 20 and 30 µg/mL) were reduced to 91 %, 61 % and 46 %, respectively of that of the concurrent negative (DMSO) control. At higher concentrations (40 and 50 µg/mL), the test substance became too toxic for the cells. In the continuous treatment group of 18 hours without metabolic activation (S9-mix), the mitotic indices of the concentrations analysed (30, 50 and 60 µg/mL ) were reduced to 93 %, 76 % and 56 %, respectively of that of the concurrent negative (DMSO) control. At higher concentrations (80 and 100 µg/mL), the test substance became too toxic for the cells.
- Genotoxicity: The test substance did not induce a statistically significant increase in the number of aberrant cells, when compared to the number of aberrant cells found in the concurrent negative (DMSO) control cultures.
HISTORICAL CONTROL DATA:
See in the field below "any other information on results incl. tables". - Conclusions:
- Under the conditions used in both independent chromosome aberration tests, the test substance ETFBO was not clastogenic for Chinese Hamster Ovary (CHO) cells.
- Executive summary:
The potential to induce structural chromosomal aberrations in vitro was investigated in a GPL-compliant study performed according to OECD test guideline 473 (In Vitro Mammalian Chromosomal Aberration Test).
Chinese Hamster Ovary (CHO) cells were tested in both the absence and presence of a Iiver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance, ETFBO was diluted in dimethylsulphoxide (DMSO). Two independent tests were performed, in the absence and the presence of S9-mix, with the following experimental conditions:
- First test:
Concentrations: 1520, 760, 380, 190, 95, 47.5, 23.8, 11.9, 5.9 and 3.0 µg/mL
Treatment / harvesting time: 4 / 18 hours (pulse treatment) in both the absence and the presence of S9-mix
- Second test:
Concentrations: 100, 80, 60, 50, 40, 30, 20, 10, 7.5, 5.0, 2.5 and 1.25 µg/mL
Treatment / harvesting time: 4 / 18 hours (pulse treatment) in the presence of S9-mix and 18 / 18 hours (continuous treatment) in the absence of S9-mix
Negative controls (DMSO) and positive controls (mitomycin C and cyclophosphamide in the absence and presence of S9-mix, respectively) were run simultaneously. Two hours before the end of the total incubation period, the cells were arrested in the metaphase stage of mitosis by the addition of colcemid. At the end of the total incubation period (18 hours at ca. 37 °C in humidified air containing ca. 5 % CO2), the cells were harvested and slides were prepared for microscopic examinations. As a measure of cytotoxicity, the mitotic index was determined. Subsequently, the cultures of selected concentrations of the test substance (selected based on mitotic index analysis), together with the negative and positive control cultures, were analysed for the induction of structural chromosomal aberrations according to the criteria recommended by Savage (1975): chromatid-type aberrations, chromosome-type aberrations and other anomalies, such as interstitial deletions, endoreduplication, polyploidy and multiple aberrations. A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cells with structural chromosomal aberrations is observed. A test substance is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberrations over the concurrent control frequencies is observed, or if a single positive test point is observed in both tests. A response is considered to be equivocal if the percentage of cells with structural chromosomal aberrations is statistically marginal higher than that of the negative control. A test substance is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points.
In both chromosomal aberration tests, the numbers of aberrant cells, found in the negative control (DMSO) cultures, were within the historical range and the positive control substances mitomycin C (in the absence of the S9-mix) and cyclophosphamide (in the presence of the S9-mix) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations. This demonstrates the validity of both chromosomal aberration tests. In both the first and second chromosomal aberration tests, the test substance ETFBO was demonstrated to be cytotoxic to the cells but did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells found in the negative control (DMSO) cultures.
Under the conditions used in both independent chromosome aberration tests, the test substance ETFBO was cytotoxic but not clastogenic for Chinese Hamster Ovary (CHO) cells.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 14 Jul.-17 Sept. 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately characterised with its purity. Therefore full validation applies.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- Draft proposal from December 2007
- Deviations:
- no
- Remarks:
- The report mentions no deviation from the draft version of the guideline available at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Human peripheral blood lymphocytes from blood samples taken by venapuncture from healthy, non-smoking males, not currently taking any medication, and collected in sterile, heparinized tubes.
- Sex of blood donors: Male.
- Number of blood donors: 2 (one donor was used for each test).
- Whether whole blood or separated lymphocytes were used: Whole blood.
- Whether blood from different donors were pooled or not: Not pooled.
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA).
No further data.
MEDIA USED
The medium for culturing the human peripheral blood lymphocytes consisted of RPMI-1640 medium (with Glutamax-I), supplemented with heat-inactivated (30 min, 56ºC) FCS (20%), penicillin (100 IU/mL medium), streptomycin (100 μg/mL medium) and phytohaemagglutinin (PHA-L; 20 Dl/mL medium). - Cytokinesis block (if used):
- Yes: Cytochalasin B (final concentration: 6 μL/mL).
- Metabolic activation:
- with and without
- Metabolic activation system:
- - Source of S9:
The S9, used in this study, was part of a batch prepared according to Ames et al. (1975) and Maron and Ames (1983). Male Wistar rats (n =12; obtained from Charles River Deutschland, Sulzfeld, Germany) were injected intraperitoneally with a single dose of Aroclor 1254 (nominal dose of 500 mg/kg body weight) in soy bean oil (20% w/v). The rats were provided with tap water and the Institute's stock diet ad libitum. Five days after the injection of Aroclor 1254 the rats were killed by CO2 asphyxiation. The livers were removed aseptically and immediately put into a cold, sterile 0.15 M KCl solution. After washing in the KCl solution, the livers were weighed, cut into pieces and homogenized in 3 volumes of 0.15 M KCl solution in a Potter-Elvehjem apparatus with a Teflon pestle. The homogenate was centrifuged for 10 minutes at 9,000 g. The supernatant, which is called S9, was collected and divided into small aliquots in sterile polypropylene vials. The vials were quickly frozen on dry ice and subsequently stored in a freezer at <-60°C.
- Method of preparation of S9 mix: Immediately before use, a S9-mix was prepared by mixing the thawed S9 with a NADPH-generating system.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4) and S9 40% (v/v). In all instances, the final concentration of the S9-mix in the culture medium was 4%.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was checked for sterility. The protein and cytochrome P-450 content of the S9 fraction were determined according to the method published by Rutten et al. (1987). The protein content of the batch was 26.6 g/L. The cytochrome P450 content of the batch was 36.0 µmol/L. The batch contained 1.35 µmol cytochrome P450 per gram protein. The sterility check of the batch resulted in 0 colonies per 100 Dl S9. The batch of S9 met all of the in-house quality criteria. - Test concentrations with justification for top dose:
- First test:
1680, 800, 400, 200, 100, 50, 25, 12.5 and 5 μg/mL.
The highest concentration of 1680 μg/mL corresponded to 10 mM which was the highest test concentration recommended in OECD 487 guideline for substances triggering no precipitate or limiting cytotoxicity. The conversion from 10 mM to 1680 μg/mL was calculated considering a molecular weight of 168 g/mol. A second in vitro micronucleus test was performed afterwards at lower concentrations (see below) due to the fact that high cytotoxicity were observed at the higher concentrations in the first test.
Second test:
800, 650, 500, 350, 200, 100, 50, 25, 10, 5 and 1 μg/mL.
In the second in vitro micronucleus test, spacing of concentrtaions was modified. The highest concentration tested was based on toxicity as observed in the first in vitro micronucleus test. - Vehicle / solvent:
- - Vehicle used: DMSO was used as solvent for the test substance, while culture medium was used as solvent for all positive control substances.
- Justification for choice of vehicle: The absence of potential of DMSO to induce micronuclei was demonstrated through assays performed in the testing laboratory (historical negative control data appended to the report summarized data in December 2008. In vitro micronucleus test was a new study in the lab at that time; only a draft guideline proposal was indeed available).
- Justification for percentage of vehicle in the final culture medium: The final DMSO concentration in the culture medium was 1% which was in accordance with the guideline recommendation indicating that organic solvents should not exceed 1% (v/v) in the final treatment medium. - Untreated negative controls:
- yes
- Remarks:
- Culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- mitomycin C
- vinblastine
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate.
- Number of independent experiments : 2 (see above in the field "Test concentrations with justification for top dose").
METHOD OF TREATMENT/ EXPOSURE AND HARVEST:
- Test substance formulation:
* First test: The test substance was dissolved in DMSO, at a stock concentration of 168 mg/mL. Thereafter, serial stock dilutions of 80, 40, 20, 10, 5, 2.5, 1.25 and 0.5 mg/mL were prepared in DMSO. Immediately after preparation the stock dilutions were clear colourless. Within 30 minutes, the stock dilutions ranging from 168 to 20 mg/mL became clear yellowish. The final concentrations of the test substance in the culture medium were 1680, 800, 400, 200, 100, 50, 25, 12.5 and 5 µg/mL.
* Second test: The test substance was dissolved in DMSO, at a stock concentration of 80 mg/mL. Thereafter, serial stock dilutions of 65, 50, 35, 20, 10, 5, 2.5, 1.0, 0.5 and 0.1 mg/mL were prepared in DMSO. Immediately after preparation the stock dilutions appeared as clear colourless solutions. Within 30 minutes, the stock dilutions ranging from 80 to 20 mg/mL became clear yellowish. The final concentrations of the test substance in the culture medium were 800, 650, 500, 350, 200, 100, 50, 25, 10, 5 and 1 µg/mL.
- Incubation period in the presence of mitogen (for both tests): In the presence of phytohaemagglutinin (PHA), aliquots of 0.5 mL of whole blood in 4.5 mL culture medium, were incubated for 48 hours at 37ºC in humidified air containing 5% CO2. The incubation was carried out in sterile screw-capped (loose) centrifuge tubes.
- Exposure conditions and duration:
* First test: The first in vitro micronucleus test was carried out both in the presence and in the absence of metabolic activation (S9-mix). After incubation, the cells were harvested by low speed centrifugation and resuspended in freshly prepared tissue culture medium without foetal calf serum (FCS) and PHA. 50 μL of the solvents (culture medium or DMSO), 50 μL of each of the dilutions of the test substance or 50 µL of the positive control substances were added to the tissue culture medium in individual culture tubes. In addition, in the presence of the S9-mix, 0.5 mL of S9-mix was added to all cultures. The total volume in each culture was 5 ml. After a 4-hour treatment period, the culture medium with the test substances and S9-mix (if any) was removed. The cells were washed twice with Phosphate-Buffered Saline (PBS; pH 7.4) and subsequently supplied with 5 mL freshly prepared culture medium enriched with PHA, FCS (20%) and Cytochalasin B (final concentration: 6 μL/mL). The cells were incubated for an additional 20 hours at 37°C in humidified air containing 5% CO2.
* Second test: The second in vitro micronucleus test was carried out in the absence of metabolic activation (S9-mix). After incubation, the cells were harvested by low speed centrifugation and resuspended in freshly prepared tissue culture medium without FCS and PHA. 50 µL of the solvents (culture medium or DMSO), 50 μL of each of the dilutions of the test substance or 50 µL of the positive control substances were added to the tissue culture medium in individual culture tubes. The total volume in each culture was 5 mL. In this second test, two groups of culture were used: one group was pulse treated and the other one was continuous treated. For the pulse treatment, the culture medium with the test substances was removed after a 4-hour treatment period. The cells were washed twice with PBS (pH 7.4) and subsequently supplied with 5 mL freshly prepared culture medium enriched with PHA, FCS (20%) and Cytochalasin B (final concentration: 6 μL/mL). The cells were incubated for an additional 20 hours at 37°C in humidified air containing 5% CO2. For the continuous treatment, the culture medium with the test substances was removed after a 20-hour treatment period. The cells were washed twice with PBS (pH 7.4) and subsequently supplied with 5 mL freshly prepared culture medium enriched with PHA, FCS (20%) and Cytochalasin B (final concentration: 6 μL/mL). The cells were incubated for an additional 28 hours at 37°C in humidified air containing 5% CO2.
- Identity and concentration of cytokinesis blocking substance and duration and period of cell exposure: Cytochalasin B at a final concentration of 6 μL/mL was used as cytokinesis blocking substance. Cells were exposed to Cytochalasin B for 20h in the first test and in the second test with pulse treatment and for 28h in the second test with continuous treatment.
- Harvest time after the end of treatment: At the end of the total incubation period, the cells in each culture were harvested and processed separately. The cells were harvested by low speed centrifugation and treated with a hypotonic solution (0.075 M potassium chloride).
- Methods of slide preparation and staining technique used including the stain used: The cells were fixed three times with a freshly prepared mixture of methanol and acetic acid, spread on clean slides and air dried. All procedures were performed at room temperature. Two slides were prepared from each selected culture of the test substance, the negative controls and positive controls. The slides were stained for 30 minutes in a 5% solution of Giemsa (filtered before use), rinsed in water, air-dried and mounted. The slides were coded by a qualified person not involved in scoring the slides, to enable "blind" scoring.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Quantitative evaluation of cytotoxicity was calculated using the Cytokinesis-Block Proliferation Index (CBPI). The CBPI indicates the average number of cell cycles per cell during the period of exposure to Cytochalasin B, and was used to calculate cell proliferation. The CBPI was determined from 500 cells per culture (in total 1000 cells per concentration) and was used to estimate the percentage of cytotoxicity by comparing values in the treated and control cultures. The CBPI, the replication index (RI) and cytotoxicity were calculated as follows:
CPBI = (no. mononucleates + 2 x no. binucleates + 3 x no. multinucleates) / Total number of cells
RI (%) = 100 x [(mean CBPI treated - 1) / (mean CBPI control - 1)]
Cytotoxicity (%) = 100 - RI
METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Number of cells spread and analysed per concentration: Two thousand binucleated cells per concentration (1000 per culture) and 2000 mononucleated cells per concentration (1000 per culture) were examined for the presence of micronuclei.
- Criteria for scoring micronucleated cells: Based on the evaluation of cytotoxicity, analysis of micronucleus formation was carried out in the negative control cultures, selected positive control cultures and in three analysable concentrations of the test substance:
* First test: 100, 200 and 400 µg/mL in the presence of S9-mix. Micronuclei formation not analysed in the absence of S9-mix due to too severe cytotoxicity.
* Second test: 100, 350 and 500 µg/mL for the pulse treatment. 25, 100 and 350 µg/mL for the continuous treatment.
N.B. The selected test substance concentrations covered a range from that producing 50 ± 5% cytotoxicity, to little or no cytotoxicity. - Evaluation criteria:
- The frequencies of binucleated and mononucleated cells with micronuclei were used for the evaluation of micronuclei induction. A response was considered to be positive if a statistically significant concentration-related or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points. A response was considered to be equivocal if the percentage of binucleated and mononucleated cells containing a micronuclei was statistically marginally higher than that of the negative control (0.05
- Statistics:
- The frequencies of micronuclei found in the ETFBO treated cultures and positive control cultures were compared with those of the concurrent solvent controls using Fisher's exact probability test (one-sided). The results were considered statistically significant when the p-value of the Fisher’s exact probability test was ≤ 0.05.
- Key result
- Species / strain:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- For further details, see in the field "Additional information on results".
- Vehicle controls validity:
- valid
- Remarks:
- The negative controls induced the expected low incidence of micronuclei.
- Untreated negative controls validity:
- valid
- Remarks:
- The negative controls induced the expected low incidence of micronuclei.
- True negative controls validity:
- other: not tested
- Positive controls validity:
- valid
- Remarks:
- Treatment with the positive control substances resulted in statistically significant increases in the number of either binucleated cells or mononucleated cells containing micronuclei, when compared to the cultures treated with the solvent control.
- Additional information on results:
- STUDY RESULTS :
- Cytotoxicity:
* First test: In the pulse treatment group with metabolic activation (S9-mix), dose-related cytotoxicity was observed, when compared to the concurrent (DMSO) control. Based on the evaluation of cytotoxicity, three dose levels (100, 200 and 400 µg/mL) of the test substance, together with the negative (culture medium and 1% DMSO) and clastogenic positive control (Cyclophosphamide; 20 µg/mL) were selected and analysed for micronucleus formation. DMSO-treated cells showed 4% cytotoxicity. The three selected dose levels of the test substance showed 11%, 17% and 50% cytotoxicity, respectively. At higher dose levels the test substance was too cytotoxic to the cells. In the pulse treatment group without metabolic activation (S9-mix), dose-related cytotoxicity was observed, when compared to the concurrent (DMSO) control. However, the Cytokinesis-Block Proliferation Index (CBPI) of both aneugenic positive controls Vinblastine (0.05 and 0.1 µg/mL) and Colchicine (0.05 and 0.1 µg/mL) showed severe cytotoxicity to the cells. Therefore, this treatment group was not analysed for micronuclei formation but discarded from the first test and repeated in the second test with lower dose levels of both aneugenic positive controls.
* Second test: In the pulse treatment group without metabolic activation (S9-mix), dose-related cytotoxicity was observed, when compared to the concurrent (DMSO) control. Based on the evaluation of cytotoxicity, three dose levels (100, 350 and 500 µg/mL) of the test substance, together with the solvent controls (culture medium and 1% DMSO), the clastogenic positive control Mitomycin C (0.4 µg/mL) and both aneugenic positive controls Vinblastine (0.0125 and 0.025 µg/mL) and Colchicine (0.02 and 0.04 µg/mL) were selected and analysed for micronucleus formation. DMSO-treated cells showed no cytotoxicity. The three selected dose levels of the test substance showed 16%, 28% and 42% cytotoxicity, respectively. At higher dose levels the test substance was too cytotoxic to the cells. In the continuous treatment group without metabolic activation (S9-mix), dose-related cytotoxicity was observed, when compared to the concurrent (DMSO) control. Based on the evaluation of cytotoxicity, three dose levels (25, 100 and 350 µg/mL) of the test substance, together with the solvent controls (culture medium and 1% DMSO), the clastogenic positive control Mitomycin C (0.1 and 0.2 µg/mL) and both aneugenic positive controls Vinblastine (0.0125 and 0.025 µg/mL) and Colchicine (0.01 and 0.02 µg/mL) were selected and analysed for micronucleus formation. DMSO-treated cells showed 4% cytotoxicity. The three selected dose levels of the test substance showed 17%, 27% and 42% cytotoxicity, respectively. At higher dose levels the test substance was too cytotoxic for the cells.
- Genotoxicity: In both the first and second in vitro micronucleus test, in both the presence and absence of a metabolic activation system (S9-mix), treatment with the test substance ETFBO did not result in a statistically significant increase in the numbers of binucleated and mononucleated cells containing micronuclei, at any of the concentrations and time points analysed, when compared to the numbers found in the concurrent solvent control cultures treated with DMSO. Treatment with the positive controls Cyclophosphamide, Vinblastine, Colchicine and Mitomycin C resulted in statistically significant increases in the number of binucleated cells or mononucleated cells containing micronuclei, when compared to the numbers found in the cultures treated with the solvent (culture medium) control. Thus the test study was considered valid.
HISTORICAL CONTROL DATA:
See in the field below "any other information on results incl. tables".Cytotoxicity and genotoxicity effects observed in both tests with ETFBO:
Treatment (µg/mL)
Mean CBPI
RI (%)
Cytotoxicity (%)
MNBN/2000 BN (%)
MNMO/2000 MO (%)
First test (pulse treatment) – With S9-mix
Culture medium (0)
1.505
-
-
9 (0.45)
4 (0.2)
DMSO (1%)
1.485
96
4
10 (0.5)
4 (0.2)
ETFBO (1680)
-
-
-
Not scored
Not scored
ETFBO (800)
1.025
5
95
Not scored
Not scored
ETFBO (400)
1.24
50
50
12 (0.6)n.s.
9 (0.45)n.s.
ETFBO (200)
1.40
83
17
8 (0.4)n.s.
9 (0.45)n.s.
ETFBO (100)
1.43
89
11
9 (0.45)n.s.
6 (0.3)n.s.
ETFBO (50)
1.45
93
7
Not scored
Not scored
ETFBO (25)
1.475
98
2
Not scored
Not scored
ETFBO (12.5)
1.485
100
0
Not scored
Not scored
ETFBO (5)
1.505
104
-4
Not scored
Not scored
Positive control: cyclophosphamide (20)
1.215
43
57
54 (2.7)***
8 (0.4)n.s.
Positive control: cyclophosphamide (10)
1.35
69
31
Not scored
Not scored
First test (pulse treatment) – Without S9-mix
Culture medium (0)
1.55
-
-
Not scored
Not scored
DMSO (1%)
1.535
97
3
Not scored
Not scored
ETFBO (1680)
-
-
-
Not scored
Not scored
ETFBO (800)
-
-
-
Not scored
Not scored
ETFBO (400)
1.27
51
49
Not scored
Not scored
ETFBO (200)
1.385
72
28
Not scored
Not scored
ETFBO (100)
1.415
78
22
Not scored
Not scored
ETFBO (50)
1.46
86
14
Not scored
Not scored
ETFBO (25)
1.485
91
9
Not scored
Not scored
ETFBO (12.5)
1.50
94
6
Not scored
Not scored
ETFBO (5)
1.535
100
0
Not scored
Not scored
Positive control: mitomycin C (0.5)
1.215
39
61
Not scored
Not scored
Positive control: mitomycin C (0.25)
1.315
57
43
Not scored
Not scored
Positive control: vinblastine (0.1)
1.01
2
98
Not scored
Not scored
Positive control: vinblastine (0.05)
1.01
2
98
Not scored
Not scored
Positive control: colchicine (0.1)
1.01
2
98
Not scored
Not scored
Positive control: colchicine (0.05)
1.075
14
86
Not scored
Not scored
Second test (pulse treatment) – Without S9-mix
Culture medium (0)
1.54
100
-
14 (0.7)
7 (0.35)
DMSO (1%)
1.59
109
-9
12 (0.6)
9 (0.45)
ETFBO (800)
-
-
-
Not scored
Not scored
ETFBO (650)
-
-
-
Not scored
Not scored
ETFBO (500)
1.34
58
42
12 (0.6)n.s.
6 (0.3)n.s.
ETFBO (350)
1.425
72
28
13 (0.65)n.s.
6 (0.3)n.s.
ETFBO (200)
1.52
88
12
Not scored
Not scored
ETFBO (100)
1.495
84
16
13 (0.65)n.s.
7 (0.35)n.s.
ETFBO (50)
1.56
95
5
Not scored
Not scored
ETFBO (25)
1.53
90
10
Not scored
Not scored
Positive control: mitomycin C (0.4)
1.345
64
36
263 (13.2)***
11 (0.55)n.s.
Positive control: vinblastine (0.025)
1.26
48
52
96 (4.8)***
121 (6.1)***
Positive control: vinblastine (0.0125)
1.465
86
14
25 (1.25)n.s.
33 (1.65)***
Positive control: vinblastine (0.00625)
1.555
103
-3
Not scored
Not scored
Positive control: vinblastine (0.00313)
1.52
96
4
Not scored
Not scored
Positive control: colchicine (0.04)
1.365
68
32
Not reported
Not reported
Positive control: colchicine (0.02)
1.50
93
7
Not scored
Not scored
Positive control: colchicine (0.01)
1.495
92
8
Not scored
Not scored
Positive control: colchicine (0.005)
1.545
101
-1
Not scored
Not scored
Second test (continuous treatment) – Without S9-mix
Culture medium (0)
1.51
100
-
13 (0.65)
4 (0.2)
DMSO (1%)
1.49
96
4
19 (0.95)
10 (0.5)
ETFBO (800)
-
-
-
Not scored
Not scored
ETFBO (650)
-
-
-
Not scored
Not scored
ETFBO (500)
-
-
-
Not scored
Not scored
ETFBO (350)
1.285
58
42
18 (0.9)n.s
15 (0.75)n.s
ETFBO (200)
1.30
61
39
Not scored
Not scored
ETFBO (100)
1.36
73
27
12 (0.6)n.s
9 (0.45)n.s
ETFBO (50)
1.37
76
24
Not scored
Not scored
ETFBO (25)
1.405
83
17
15 (0.75)n.s
9 (0.45)n.s
ETFBO (10)
1.46
94
6
Not scored
Not scored
ETFBO (5)
1.415
85
15
Not scored
Not scored
ETFBO (1)
1.42
86
14
Not scored
Not scored
Positive control: mitomycin C (0.2)
1.315
62
38
857 (42.9)***
60 (3.0)***
Positive control: mitomycin C (0.1)
1.39
76
24
457 (22.9)***
40 (2.0)***
Positive control: vinblastine (0.025)
1.30
59
41
48 (2.4)***
87 (4.35)***
Positive control: vinblastine (0.0125)
1.485
95
5
14 (0.7)n.s
38 (1.9)***
Positive control: vinblastine (0.00625)
1.44
86
14
Not scored
Not scored
Positive control: vinblastine (0.00313)
1.44
86
14
Not scored
Not scored
Positive control: colchicine (0.04)
1.08
16
84
Not scored
Not scored
Positive control: colchicine (0.02)
1.305
60
40
14 (0.7)n.s
51 (2.6)***
Positive control: colchicine (0.01)
1.445
87
13
15 (0.75)n.s
16 (0.8)**
Positive control: colchicine (0.005)
1.49
96
4
Not scored
Not scored
Fisher's exact probability test (one-sided): n.s. (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001)
Cytokinesis-Block Proliferation Index, CBPI = (no. mononucleates + 2 x no. binucleates + 3 x no. multinucleates) / Total no. of cells
Replication index, RI (%) = 100 x [(mean CBPI treated - 1) / (mean CBPI control - 1)]
Cytotoxicity (%) = 100 - RI
MNBN/2000 BN = Number of micronucleus scored in 2000 binucleated cells
MNMO/2000 MO = Number of micronucleus scored in 2000 mononucleated cells
Historical negative controls (culture medium and DMSO): summarized data (December 2008):
Culture1)/ Treatment2)/ Recovery3)
(h)
With or without S9-mix
Vehicle
% of binucleated
cells containing
micronuclei
Number of treatment groups performed
24 / 4 / 44
+ S9-mix
Medium
DMSO
0.4
0.6
1
1
48 / 4 / 44
+ S9-mix
Medium
DMSO
0.6
0.6
1
1
24 / 20 / 28
- S9-mix
Medium
DMSO
0.6
0.6
1
1
48 / 20 / 28
- S9-mix
Medium
DMSO
0.8
0.8
1
1
1) Culture time (h) prior to treatment in the presence of phytohaemagglutinin
2) Treatment period (h)
3) Recovery time (h) after the end of treatment in the presence of phytohaemagglutinin and cytochalasin B
N.B. During the first test conducted as a pulse treatment (48/4/20h) in the presence of S9-mix in the context of the present study, 0.45% and 0.5% binucleated cells containing micronuclei were recorded in the treatments exposed to culture medium and DMSO, respectively. During the second test conducted as a pulse treatment (48/4/20h) in the absence of S9-mix, 0.7% and 0.6% binucleated cells containing micronuclei were recorded in the treatment exposed to culture medium and DMSO, respectively. During the second test conducted as a continuous treatment (48/20/28h) in the absence of S9-mix, 0.65% and 0.95% binucleated cells containing micronuclei were recorded in the treatment exposed to culture medium and DMSO, respectively (see in the first table above). The comparison with the historical negative control data reported in the table immediately above is not easy because different culture/treatment/recovery durations were tested and also because only one assay composed the historical database for each combination negative control / duration; certainly due to the fact that the guideline was pretty new at the time of the study (only a draft guideline proposal was indeed available). Nevertheless, it can be considered that culture medium and DMSO induced the expected low incidence of micronuclei.
Historical positive controls: summarized data (December 2008):
Culture1)/ Treatment2)/ Recovery3)
(h)
Dose level (µg/mL)
% of binucleated
cells containing
micronuclei
Number of treatment groups performed
Indirect acting clastogen Cyclophosphamide – With S9-mix
24 / 4 / 44
5
10
15
3.6
4.0
11.0
1
1
1
48 / 4 / 44
5
10
15
4.6
9.0
14.0
1
1
1
Direct acting clastogen Mitomycin C – Without S9-mix
24 / 20 / 28
0.25
0.125
42.4
23.6
1
1
48 / 20 / 28
0.25
0.125
42.6
26.0
1
1
Aneugenic compound Vinblastine – Without S9-mix
24 / 20 / 28
0.25
0.125
0.0625
0.0313
12.4
11.6
6.2
7.0
1
1
1
1
48 / 20 / 28
0.0625
0.0313
8.6
6.4
1
1
1)Culture time (h) prior to treatment in the presence of phytohaemagglutinin
2)Treatment period (h)
3)Recovery time (h) after the end of treatment in the presence of phytohaemagglutinin and cytochalasin B
- Conclusions:
- Under the conditions used in both independent in vitro micronucleus tests, the test substance ETFBO was neither clastogenic nor aneugenic to cultured human lymphocytes.
- Executive summary:
The potential to induce micronuclei in vitro was investigated in a GPL-compliant study performed according to OECD test guideline 487 (In Vitro Mammalian Cell Micronucleus Test).
Human peripheral blood lymphocytes were tested in both the absence and presence of a Iiver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The test substance, ETFBO was diluted in dimethylsulphoxide (DMSO). Two independent tests were performed, with the following experimental conditions:
- First test:
* Concentrations:
1680, 800, 400, 200, 100, 50, 25, 12.5 and 5 μg/mL
* Culture / Treatment / Recovery time:
48 / 4 / 20 hours (pulse treatment) in both the absence and the presence of S9-mix
- Second test:
* Concentrations:
800, 650, 500, 350, 200, 100, 50, 25, 10, 5 and 1 μg/mL
* Culture / Treatment / Recovery time:
48 / 4 / 20 hours (pulse treatment) in the absence of S9-mix
48 / 20 / 28 hours (continuous treatment) in the absence of S9-mix
Negative controls (DMSO and culture medium) and positive controls (in the presence of S9-mix: indirect acting clastogen Cyclophosphamide, in the absence of S9-mix: direct acting clastogen Mitomycin C and aneugenic compound Vinblastine) were run simultaneously. In both tests and under all conditions, incubation lasted for 48 hours and occurred in the presence of the mitogen phytohaemagglutinin (PHA). After the appropriate treatment time (4 or 20 hours for pulse and continuous treatments, respectively), cells were incubated for an additional 20-hour (pulse treatment) or 28-hour (continuous treatment) period in the presence of the cytokinesis blocking substance Cytochalasin B. At the end of the total incubation period, the cells were harvested and slides were prepared for microscopic examinations. As a measure of cytotoxicity, the Cytokinesis-Block Proliferation Index (CBPI) was determined. Subsequently, the cultures of selected concentrations of the test substance (selected based on cytotoxicity percentage), together with the negative and positive control cultures, were analysed for the induction of micronucleus. The frequencies of binucleated and mononucleated cells with micronuclei were used for the evaluation of micronuclei induction. A response was considered to be positive if a statistically significant concentration-related or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points. A response was considered to be equivocal if the percentage of binucleated and mononucleated cells containing a micronuclei was statistically marginally higher than that of the negative control. A test substance was considered to be negative if it produces neither a statistically significant concentration-related or reproducible statistically significant increase in the number of binucleated and mononucleated cells containing micronuclei, at any of the test points.
The study was considered valid because (i) the selected clastogenic positive controls gave a statistically significant increase in the number of binucleated cells containing micronuclei, (ii) the selected aneugenic positive controls gave a statistically significant increase in the number of mononucleated cells containing micronuclei, (iii) the negative controls induced the expected low incidence of micronuclei. In both the first and second in vitro micronucleus tests, in both the presence and absence of a metabolic activation system (S9-mix), the test substance ETFBO was demonstrated to be cytotoxic to the cells, but did not induce statistically significant increase in the numbers of binucleated and mononucleated cells containing micronuclei, at any of the concentrations and time points analysed, when compared to the numbers found in the concurrent solvent control cultures treated with DMSO.
Under the conditions used in both independent in vitro micronucleus tests, the test substance ETFBO was cytotoxic, but neither clastogenic nor aneugenic to cultured human lymphocytes.
Referenceopen allclose all
Number of revertants counted in the bacterial reverse mutation test with ETFBO:
Figures presented in the table below are average numbers of revertants per plate (standard deviations).
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
First test |
||||||||||
Negative control |
22 (6) |
15 (4) |
16 (5) |
11 (6) |
26 (5) |
43 (13) |
126 (19) |
125 (10) |
30 (10) |
34 (8) |
62 µg/plate |
19 (5) |
17 (3) |
11 (6) |
13 (3) |
26 (4) |
40 (9) |
116 (20) |
134 (19) |
40 (9) |
38 (4) |
185 µg/plate |
24A (2) |
21A (3) |
11A (3) |
13A (7) |
29A (3) |
36A (8) |
80A (21) |
77A (9) |
13B (5) |
20A (3) |
556 µg/plate |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
1667 µg/plate |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
5000 µg/plate |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
pp |
Positive control |
605 (12) |
356 (55) |
1135 (448) |
272 (19) |
1499 (70) |
708 (124) |
738 (39) |
1794 (111) |
234 (32) |
1812 (81) |
Second test |
||||||||||
Negative control |
19 (3) |
13 (2) |
6 (1) |
9 (4) |
31 (8) |
51 (4) |
110 (15) |
117 (8) |
19 (5) |
33 (9) |
16 µg/plate |
20 (3) |
19 (4) |
14 (2) |
11 (1) |
25 (3) |
43 (12) |
123 (5) |
114 (11) |
21 (6) |
32 (5) |
31.25 µg/plate |
17 (1) |
18 (2) |
8 (2) |
8 (1) |
28 (3) |
45 (6) |
112 (16) |
124 (10) |
21 (5) |
27 (4) |
62.5 µg/plate |
22 (3) |
11 (3) |
7 (3) |
11 (3) |
28 (6) |
50 (3) |
106 (15) |
141 (20) |
25 (2) |
36 (9) |
125 µg/plate |
16 (5) |
12 (0) |
11A (1) |
13 (6) |
25A (4) |
48A (6) |
108 (7) |
116 (4) |
20A (12) |
31 (9) |
250 µg/plate |
17A (3) |
17A (4) |
10A (2) |
10A (4) |
25A (8) |
41A (5) |
91A (26) |
113A (20) |
15A (5) |
23A (1) |
500 µg/plate |
10B (3) |
16B (7) |
2C (1) |
1C (2) |
8C (7) |
27C (10) |
8C (2) |
12B (4) |
11B (1) |
17B (3) |
Positive control |
655 (98) |
309 (16) |
1464 (49) |
298 (46) |
1472 (60) |
779 (146) |
808 (74) |
1968 (59) |
300 (43) |
1382 (303) |
pp = pinpoint colonies
A= slightly less dense background lawn of bacterial growth
B= less dense background lawn of bacterial growth
C= less dense background lawn of bacterial growth and pinpoint colonies
Overview of historical positive control data from studies between May 2002 and March 2005:
Strain |
Compound |
Mutation ratio |
|||
Mean |
Standard deviation |
Range |
Number of assays |
||
Without S9-mix |
|||||
TA1535 |
NaN3 1µg/plate |
31 |
37 |
13-297 |
(57) |
TA1537 |
9-AA 80µg/plate |
191 |
76 |
49-350 |
(57) |
TA98 |
2-NF 2µg/plate |
49 |
17 |
16-98 |
(66) |
TA100 |
NaN3 1µg/plate |
5 |
1 |
3-8 |
(60) |
WP2 uvrA |
ENU 100µg/plate |
6 |
3 |
3-9 |
(55) |
With S9-mix |
|||||
TA1535 |
2-AA 2 µg/plate |
28 |
9 |
11-61 |
(57) |
TA1537 |
BP 4 µg/plate |
13 |
5 |
6-35 |
(57) |
TA98 |
2-AA 2µg/plate |
24 |
11 |
9-80 |
(67) |
TA100 |
2-AA 2µg/plate |
15 |
3 |
8-22 |
(60) |
WP2 uvrA |
2-AA 80µg/plate |
36 |
8 |
20-57 |
(55) |
NaN3 = natrium azide
ENU = N-nitroso N-ethylurea
2-AA = 2-aminoanthracene
9-AA = 9-aminoacridine
BP = benzo(a)pyrene
2-NF = 2-nitrofluorene
Overview of historical negative control (DMSO) data from studies between May 2002 and March 2005:
Strain |
Number of revertants |
||
Mean |
Standard deviation |
Number of assays |
|
Without S9-mix |
|||
TA1535 |
26 |
5 |
20 |
TA1537 |
18 |
5 |
20 |
TA98 |
38 |
5 |
28 |
TA100 |
172 |
27 |
24 |
WP2 uvrA |
38 |
7 |
20 |
With S9-mix |
|||
TA1535 |
20 |
5 |
20 |
TA1537 |
23 |
5 |
20 |
TA98 |
58 |
8 |
28 |
TA100 |
172 |
29 |
20 |
WP2 uvrA |
42 |
6 |
20 |
Results of the two independent chromosome aberration tests with ETFBO:
Treatment / harvest time (h) |
Dose level (µg/mL) |
Cell showing structural chromosome aberrations (%) |
Relative Mitotic Index (%) |
Cell showing numerical chromosome aberrations (%) |
First test – With S9-mix |
||||
4 / 18 |
Negative control |
0.5(3) |
100 |
0.0 |
5.9 |
0.0- |
86 |
0.0 |
|
11.9 |
0.5-(3) |
73 |
0.0 |
|
23.8 |
0.0- |
46 |
0.0 |
|
Positive control |
23.5 ***(1,2,3,4) |
38 |
0.0 |
|
First test – Without S9-mix |
||||
4 / 18 |
Negative control |
0.0 |
100 |
0.0 |
11.9 |
0.0 - |
102 |
0.0 |
|
23.8 |
0.5 - (1) |
109 |
0.0 |
|
47.5 |
0.0 - |
97 |
0.0 |
|
Positive control |
21.0 ***(1,2,3,4) |
84 |
0.0 |
|
Second test – With S9-mix |
||||
4 / 18 |
Negative control |
0.5 (2) |
100 |
0.0 |
10.0 |
2.0 - (1,2) |
91 |
0.0 |
|
20.0 |
1.0 - (1) |
61 |
0.0 |
|
30.0 |
1.0 - (1) |
46 |
0.0 |
|
Positive control |
36.0 *** (1,2) |
41 |
0.0 |
|
Second test – Without S9-mix |
||||
18 / 18 |
Negative control |
0.5 (1) |
100 |
0.0 |
30.0 |
0.5 - (1) |
93 |
0.0 |
|
50.0 |
0.0 - |
74 |
0.0 |
|
60.0 |
1.5 - (1) |
56 |
0.0 |
|
Positive control |
33.5 *** (1,2,4) |
75 |
0.0 |
|
Fisher's exact probability test (two-sided): - p > 0.05, *** p ≤ 0.00 1
Types of structural chromosome aberrations:
(1)chromatid break
(2)chromatid exchange
(3)chromosome break
(4)chromosome exchange
Overview of historical negative control (DMSO) data from studies between 2000 and 2003:
Treatment / harvest time (h) |
% of cells with aberrations |
|||
Mean |
Standard deviation |
Range |
Number of treatment groups performed |
|
Without S9-mix |
||||
4 / 18 |
0.8 |
1.1 |
0.0 – 4.5 |
20 |
18 / 18 |
0.6 |
0.8 |
0.0 – 3.5 |
31 |
With S9-mix |
||||
4 / 18 |
0.8 |
1.0 |
0.0 – 4.5 |
33 |
Overview of historical positive control data from studies between 2000 and 2003:
Treatment / harvest time (h) |
Compound |
% of cells with aberrations |
|||
Mean |
Standard deviation |
Range |
Number of treatment groups performed |
||
Without S9-mix |
|||||
4 / 18 |
Mitomycin C (0.1 µg/mL) |
28.0 |
7.1 |
15.0 – 40.5 |
43 |
18 / 18 |
Mitomycin C (0.05 µg/mL) |
30.0 |
6.5 |
15.5 – 43.5 |
55 |
18 / 18 |
Mitomycin C (0.025 µg/mL) |
15.4 |
5.0 |
4.0 – 21.0 |
11 |
With S9-mix |
|||||
4 / 18 |
Cyclophosphamide (3 µg/mL) |
21.0 |
9.7 |
5.5 – 47.0 |
56 |
4 / 18 |
Cyclophosphamide (3.75 µg/mL) |
29.7 |
11.4 |
6.5 – 45.5 |
13 |
4 / 18 |
Cyclophosphamide (5 µg/mL) |
33.0 |
2.0 |
31.5 – 34.5 |
2 |
N.B. During both tests performed in the presence of S9-mix in the context of the present study, 23.5% and 36% cells showing structural chromosome aberrations were recorded in the treatment exposed to cyclophosphamide (5 µg/mL; see in the first table above). This appears to be outside the historical range reported in the table immediately above for cyclophosphamide at 5 µg/mL (i.e. 31.5 to 34.5%). However, it has to be noticed that only two assays composed the historical database for this positive control at this concentration; explaining the narrowness of the range. As it can be seen for the same positive control at other concentrations (3 and 3.75 µg/mL), when performing more assays, the historical range becomes wider. As a result, it can be considered that cyclophosphamide induced an expected statistically significant increases in the incidence of structural chromosomal aberrations and that the fact to be outside the historical range is only due to the low number of assays covered and has no influence on the integrity of the study results.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro: Three GPL-compliant studies performed according to OECD test guideline 471 (Bacterial Reverse Mutation Assay, Ames test), OECD test guideline 473 (In Vitro Mammalian Chromosome Aberration Test) and OECD test guideline 487 (In Vitro Mammalian Cell Micronucleus Test) are available. Despite restrictions due to incomplete test item identification (i.e. unspecified cis-trans isomery), OECD 471 and OECD 473 studies are considered as reliable with restriction and their results are included as part of the weight-of-evidence approach used to cover this endpoint. OECD 487 study is considered as fully reliable and the result is also included as part of the weight-of-evidence approach used to cover this endpoint.
Justification for classification or non-classification
The three available in vitro studies allows concluding that ETFBO does not require a classification for genetic toxicity.
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