Registration Dossier
Registration Dossier
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EC number: 915-687-0 | CAS number: 1065336-91-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial and mammalian cell gene mutation tests revealed no mutagenic potential. The chromosome aberration test in vitro resulted in a clastogenic response which could not be confirmed in the in vivo assay.
A structurally related source substance was not mutagenic in bacterial and mammalian cells and did not induce clastogenic effects in a chromosome aberration test with cultured human peripheral lymphocytes with or without metabolic activation.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 Feb 2010 - 30 Jun 2010
- 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)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL) and 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 1st Experiment (without light protection)
(without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL)
(with S9 mix: 0; 312.5; 625; 1 250; 2 500; 3750; 5000 μg/mL)
2nd Experiment (without light protection)
without S9 mix: 0; (5); (10); (20); 40; 60; 80 μg/mL
with S9 mix: 0; (78.1); (156.3); 312.5; 625; 1250; (2500); (3750) μg/mL
3rd Experiment (under light protection)
without S9 mix: 0; 6.3; 12.5; 25; (50); (100), (200) μg/mL
with S9 mix: 0; 156.3; 312.5; 625; (1250); (2500); (5000) μg/mL
(test groups in brackets were not evaluated) - Vehicle / solvent:
- acetone 1% (v/v)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- EMS: 500 μg/mL; CPP: 0.5 μg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h (both with and without S9)
- Expression time (cells in growth medium): 14 h (both with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h (both with and without S9)
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2
NUMBER OF REPLICATIONS: All cultures were prepared in duplicate.
NUMBER OF CELLS EVALUATED: 100 metaphases per culture were evaluated for structural chromosome aberrations. Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; a mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in all cytogenetic experiments.
OTHER EXAMINATIONS:
Aneuploid and polyploid cells were recorded separately. - Evaluation criteria:
- As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 – 22 chromosomes, they were analyzed for structural chromosome aberrations.
The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range. - Statistics:
- The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the main experiments, test substance precipitation in culture medium at the end of exposure period was observed from 625 μg/mL onward only in the presence of S9 mix in all experimental parts.
RANGE-FINDING/SCREENING STUDIES:
In the pretest performed without light protection the pH was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in the vehicle acetone was observed up to the highest required concentration of 5000 μg/mL (stock solution). In culture medium test substance precipitation occurred at 625 μg/mL and above 4 hours after start of treatment in the absence and the presence of S9 mix as well as at 18 hours continuous treatment in the absence of S9 mix. At these concentrations an oily film of the test substance was observed on surface of the culture medium.
After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced cell numbers of below 50% of control was observed at 78.1 μg/mL and above. In addition, in the presence of S9 mix, clearly reduced cell numbers were observed after treatment with 5000 μg/mL. Besides, in the pretest with 18 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 156.3 μg/mL.
COMPARISON WITH HISTORICAL CONTROL DATA:
Second experiment: In the presence of metabolic activation after 4 hours treatment a statistically significant increase in the number of chromosomally damaged cells was observed at the highest scorable concentration of 1250 μg/mL (18% aberrant metaphases excl. gaps). This value clearly exceeded our historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps).
Third experiment: In the absence and presence of S9 mix a statistically significant increase in the number of aberrant metaphases was observed at all concentrations scored for cytogenetic damage performed under light protection conditions. In both experimental parts most values exceeded our historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps).
CELL MORPHOLOGY
Cell attachment was influenced from 40.0 μg/mL onward in the 1st or 2nd Experiment without S9 mix. When applying light protection in the 3rd Experiment the cell attachment was slightly reduced from 12.5 μg/mL onward in the absence of S9 mix. After the addition of S9 mix cell morphology was influenced from about 625 μg/mL onward in all experiments performed with or without light exclusion.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix 4 hours after treatment in the 2nd and 3rd Experiment, no suppression of the mitotic activity was observed at any dose analyzed for chromosomal aberrations. In the 2nd Experiment in the absence of metabolic activation after 4 hours treatment up to 80 μg/mL no reduction of mitotic activity was obtained. In the 3rd Experiment in the absence of metabolic activation the slides from 50 μg/mL onward were not scorable due to low metaphase numbers. Besides, in the presence of S9 mix in the 2nd and 3rd Experiment after 4 hours treatment the concentrations showing clearly reduced mitotic indices were not scorable for cytogenetic damage due to low metaphase numbers and/or poor metaphase quality (from 2 500 μg/mL or 1 250 μg/mL onward, respectively). - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Under the experimental conditions chosen here, the conclusion is drawn that the test article is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence of a metabolizing system. During the experimental phase of this study the lab was informed by the sponsor that the test substance is an UV-curing agent. Therefore, it is light sensitive and has to be stored and handled protected from light. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested and the test groups in bold type were evaluated:
1st Experiment (without light protection)
4-hour exposure, 18-hour sampling time, without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix: 0; 312.5; 625; 1250; 2500; 3750; 5000 μg/mL
The 1st Experiment with and without S9 mix had to be repeated due to severe cytotoxicity (strongly reduced mitotic rates) and partly poor metaphase quality.
2nd Experiment (without light protection)
4-hour exposure, 18-hour sampling time, without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix: 0; 78.1; 156.3; 312.5; 625; 1250; 2500; 3750 μg/mL
The 2nd Experiment with and without S9 mix had to be repeated due to an update on storage conditions and handling instructions indicating protection from light.
3rd Experiment (under light protection)
4-hour exposure, 18-hour sampling time, without S9 mix: 0; 6.3; 12.5; 25; 50; 100, 200 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix: 0; 156.3; 312.5; 625; 1250; 2500; 5000 μg/mL
A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures and the concentrations 1250 μg/mL and 625 μg/mL in the 2nd and 3rd Experiment, respectively, where only 50 metaphases were scored due to clearly increased aberration rates. The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after 4 hours exposure without light protection in the presence of metabolic activation. in a single experiment (2nd Experiment). In the 3rd Experiment under light protection a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps was obtained after 4 hours exposure either without S9 mix or after adding a metabolizing system (see Table 1, page 14 – 15). The observation of clastogenicity in the 2nd Experiment was confirmed in the 3rd Experiment (performed under light protection conditions). Therefore, it can be assumed that the toxic effect obtained in the 2nd Experiment was not induced by light absorption. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the test article is considered to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March 30, 2010 - September 10, 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % neomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- without S9: 0.6 - 160 microg/mL
with S9: 20 - 320 microg/mL - Vehicle / solvent:
- tetrahydrofuran (THF, not excceding 0.5% (v/v) in the culture medium
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- THF
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- EMS: 0.150 mg/mL; DMBA: 1.1 μg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 days
SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
STAIN: 10 % methylene blue in 0.01 % KOH solution
NUMBER OF REPLICATIONS: two parallel cultures for each concentration
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 106 cells found in the solvent con¬trols falls within the laboratory historical control data from 2006 - 2008
b) the positive control substances should produce a significant increase in mutant colony frequencies.
c) the cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- for details see below
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH
- Effects of osmolality: There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration in the pre-test on toxicity (2600 μg/mL) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 476. Strong cytotoxic effects occurred at 162.5 μg/mL without metabolic activation and at 325 μg/mL and above with metabolic activation following 4 hours treatment. After continuous treatment (24 hours), solely performed without metabolic activation, relevant toxic effects were noted at 81.3 μg/mL and above. The test medium was checked for precipitation or turbidity at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye at 2600 μg/mL after 4 and 24 hours treatment in the absence of metabolic activation. In the presence of metabolic activation precipitation occurred after 4 hours treatment at 1300 μg/mL treatment at 1300 μg/mL and above. In the pre-experiment the two highest concentrations were neutralised with hydrochloric acid. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
COMPARISON WITH HISTORICAL CONTROL DATA:
In the main experiments (with and without S9 mix) the range of the solvent controls was from 11.3 up to 35.1 mutants per 106 cells; the range of the groups treated with the test item was from 3.4 up to 46.1 mutants per 106 cells. The highest solvent control slightly exceeded the historical range (35.1 versus 31.5 colonies per 106 cells). This deviation was considered irrelevant since it was very minor and the solvent control of the parallel culture remained well within the historical range.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50% in at least one of the parallel cultures occurred in the first experiment at 20.0 μg/mL and above without metabolic activation and at 160 μg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 80.0 μg/mL without and 240 μg/mL with metabolic activation. In the additional experiment IIA, cytotoxicity was noted at 80.0 μg/mL and above. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I was covered with and without metabolic activation. - Conclusions:
- Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
- Executive summary:
The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster following OECD guideline 476. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. A third experiment without metabolic activation was performed since the recommended cytotoxic range of approximately 10-20% was not covered in the second experiment without metabolic activation. The third experiment, designated experiment IIA, was performed with a treatment time of 24 hours. The cell cultures were evaluated at the following concentrations: Experiment I: without S9 mix: 1.3; 2.5; 5.0; 10.0; and 20.0 μg/mL; with S9 mix: 20.0; 40.0; 80.0; and 160.0 μg/mL Experiment II: without S9 mix: 5.0; 10.0; 20.0; 40.0; and 80.0 μg/mL; with S9 mix: 40.0; 80.0; 120.0; 180.0; and 240.0 μg/mL Experiment IIA: without S9 mix: 10.0; 20.0; 40.0; and 80.0 μg/mL The highest concentration of the main experiments was limited by the cytotoxicity of the test item. Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50% occurred in the first experiment at 20.0 µg/mL and above without metabolic activation and at 160 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 80.0 µg/mL without and 240 µg/mL with metabolic activation. In the additional experiment IIA, cytotoxicity was noted at 80.0 µg/mL and above. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I was covered with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor did not reach the threshold of three times the mutation frequency of the corresponding solvent controls in any of the experimental parts. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation and in the first culture of experiment IIA without metabolic activation. These increases however, were judged as irrelevant since the induction factor did not reach the threshold discussed above. In the main experiments (with and without S9 mix) the range of the solvent controls was from 11.3 up to 35.1 mutants per 106 cells; the range of the groups treated with the test item was from 3.4 up to 46.1 mutants per 106 cells. The highest solvent control slightly exceeded the historical range (35.1 versus 31.5 colonies per 106 cells). This deviation was considered irrelevant since it was very minor and the solvent control of the parallel culture remained well within the historical range. EMS (0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
- 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:
- From Jul. 16, 1980 to Sep. 8, 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- No independent repeat test conducted.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively.
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- Toxicity test (performed on strain TA 100): 0.0001, 0.001, 0.01, 0.1, 1, 10, 50, 100, 500, 1000, and 2000 µg/0.1 mL.
Experiment (performed on all strains): 5, 10, 50, 100, 500, 1000, and 5000 µg/0.1 mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: None provided.
Method for suspension of refractory substances: Ultrasonication - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: See Table 1
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 - 55 hours at 37 degrees C.
NUMBER OF REPLICATIONS: 3 Petri dishes/strain/group.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- When the colonies had been counted, the arithmetic mean was calculated. A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
- Statistics:
- When the colonies had been counted, the arithmetic mean was calculated. Statistical analysis was not performed.
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 500, 1,000, and 5,000 µg/0.1mL, the substance precipitated in soft agar. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- No evidence for a mutagenic effect was obtained in Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E.coli strain WP2 uvr A treated with up to 5000 µg/plate in experiments with and without microsomal activa¬tion.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 200
- 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)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Merck), penicillin/streptomycin (50 U/ml and 50 ng/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, the Netherlands).
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and beta-naphthoflavone induced rat liver S9-mix
- Test concentrations with justification for top dose:
- Dose-range finding test: 10-1000 µg/mL (with S9-mix), 10-3300 µg/mL (without S9-mix)
First cytogenetic assay: 50-750 µg/ml (with and without S9-mix)
Second cytogenetic assay: 50-1000 µg/mL (without S9-mix), 50-750 µg/mL (with S9-mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Mitomycim C without S9-mix, Cyclophosphamide with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3h (with and without S9), 24 and 48h (without S9)
- Expression time: After the 3h exposure, the cells were washed and incubated for another 20-22 h (24 h fixation time) or 44-46 h (48 h fixation time). Cells treated for 24 h and 48 h in the absence of S9-mix were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium)
STAIN (for cytogenetic assays): 5% (v/v) Giemsa (Merck) solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-responserelationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. - Statistics:
- Chi-square test, one-sided, p< 0.05
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics. - Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: variable, from 500, 600 or 700 µg/ml and up
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occasionally observed at 750 µg/ml and higher
RANGE-FINDING/SCREENING STUDIES:
At a concentration of 1000 µg/ml the test article precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 10, 33, 100, 333 and 1000 µg/ml culture medium with and without S9-mix. At the 24 h and 48 h continuous exposure time blood cultures were treated with 10, 33, 100, 333, 1000 and 3330 µg/ml culture medium without S9-mix. The test substance was tested beyond the limit of solubility to obtain adequate toxicity data.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The observed cytotoxicity was variable, sometimes starting at around 400 µg/ml, in other occasions not before 700 µg/ml. The variable cytotoxicity observed can be caused by the sensitivity of the different donors used for the different experiments. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The test article was not clastogenic in the presence or absence of metabolic activation
Referenceopen allclose all
The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after 4 hours exposure without light protection in the presence of metabolic activation in a single experiment (2nd Experiment). In the 3rd Experiment under light protection a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps was obtained after 4 hours exposure either without S9 mix or after adding a metabolizing system. The observation of clastogenicity in the 2nd Experiment was confirmed in the 3rd Experiment (performed under light protection conditions). Therefore, it can be assumed that the toxic effect obtained in the 2nd Experiment was not induced by light absorption. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either.
Summary table – experimental parts without S9 mix
| Exp. | Schedule | Test groups | S9 mix | P | Genotoxicity | Cytotoxicity* | ||||
| Exposure/ preparation period | Aberrant cells [%] | Polyploidy cells [%] | ||||||||
| Incl. gaps# | Excl. gaps# | With exchanges | Cell no. [%] | Mitotic index [%] | ||||||
| 2 | 4/18 h | Vehicle control1 | - | - | 7 | 3 | 0.5 | 0 | 100 | 100 |
| 5.0 mg/mL | - | - | n.d. | n.d. | n.d. | n.d. | 98 | n.d. | ||
| 10.0 mg/mL | - | - | n.d. | n.d. | n.d. | n.d. | 85 | n.d. | ||
| 20.0 mg/mL | - | - | n.d. | n.d. | n.d. | n.d. | 81.8 | n.d. | ||
| 40.0 mg/mL | - | - | 5 | 3.5 | 1 | 0 | 82.9 | 111.6 | ||
| 60.0 mg/mL | - | - | 4.5 | 3.5 | 1 | 0 | 81.8 | 105.5 | ||
| 80.0 mg/mL | - | - | 7 | 5.5 | 3.5 | 0 | 38.7 | 103.4 | ||
| Positive control2x | - | - | 21.0s | 18.0s | 10.0s | 0 | n.t. | 103.4 | ||
| Performance under light protection | ||||||||||
| 2 | 4/18 h | Vehicle control1 | - | - | 4 | 1.5 | 0.5 | 0 | 100 | 100 |
| 6.3 mg/mL | - | - | 8.5s | 5.5s | 3 | 1 | 74.8 | 89.4 | ||
| 12.5 mg/mL | - | - | 13.5s | 9.5s | 7.5s | 0.5 | 47.6 | 105.3 | ||
| 25.0 mg/mL | - | - | 14.5s | 10.0s | 6.5s | 0 | 46.5 | 104.6 | ||
| 50.0 mg/mL | - | - | n.s. | n.s. | n.s. | n.s. | 4.1 | n.s. | ||
| 100.0 mg/mL | - | - | n.s. | n.s. | n.s. | n.s. | 3.2 | n.s. | ||
| 200.0 mg/mL | - | - | n.s. | n.s. | n.s. | n.s. | 4.8 | n.s. | ||
| Positive control2x | - | - | 20.0s | 17.0s | 10.0s | 0 | n.t. | 82.8 | ||
Summary table – experimental parts with S9 mix
| Exp. | Schedule | Test groups | S9 mix | P | Genotoxicity | Cytotoxicity* | ||||
| Exposure/ preparation period | Aberrant cells [%] | Polyploidy cells [%] | ||||||||
| Incl. gaps# | Excl. gaps# | With exchanges | Cell no. [%] | Mitotic index [%] | ||||||
| 2 | 4/18 h | Vehicle control1 | + | - | 5 | 3.5 | 1.5 | 0 | 100 | 100 |
| 78.1 mg/mL | + | - | n.d. | n.d. | n.d. | n.d. | 88.9 | n.d. | ||
| 156.3 mg/mL | + | - | n.d. | n.d. | n.d. | n.d. | 89.8 | n.d. | ||
| 312.5 mg/mL | + | - | 7.5 | 5 | 3.5 | 0 | 78.5 | 103 | ||
| 625.0 mg/mL | + | - | 6 | 4.5 | 3 | 0 | 78.5 | 79.2 | ||
| 1250.0 mg/mLx | + | + | 18.0s | 18.0s | 16.0s | 0 | 55.2 | 81.7 | ||
| 2500.0 mg/mL | + | + | n.s. | n.s. | n.s. | n.s. | 56.7 | 29.2 | ||
| 3750.0 mg/mL | + | + | n.s. | n.s. | n.s. | n.s. | 27.4 | n.s. | ||
| Positive control3x | + | - | 27.0s | 25.0s | 10.0s | 0 | n.t. | 60.4 | ||
| Performance under light protection | ||||||||||
| 2 | 4/18 h | Vehicle control1 | + | - | 4.5 | 2 | 0.5 | 0 | 100 | 100 |
| 156.3 mg/mL | + | - | 11.5s | 9.0s | 4.0s | 0.5 | 87.1 | 84.8 | ||
| 312.5 mg/mL | + | - | 13.0s | 9.0s | 4.0s | 0.5 | 83 | 99 | ||
| 625.0 mg/mLx | + | + | 31.0s | 30.0s | 23.0s | 0 | 73 | 85.3 | ||
| 1250.0 mg/mL | + | + | n.s. | n.s. | n.s. | n.s. | 48.8 | 40.6 | ||
| 2500.0 mg/mL | + | + | n.s. | n.s. | n.s. | n.s. | 51.2 | n.s. | ||
| 500.0 mg/mL | + | + | n.s. | n.s. | n.s. | n.s. | 41.4 | n.s. | ||
| Positive control3x | + | - | 26.0s | 22.0s | 12.0s | 0 | n.t. | 77.7 | ||
P Precipitation occured at the end of exposure period
* Relative values compared with the respective vehicle control
# Inclusive cells carrying exchanges
s Aberration frequency statistically significant higher than corresponding control values
x Evaluation of a sample of 100 metaphase only due to strong clastogenicity
1 Acetone 1% (v/v)
2 EMS500 μg/mL
3 CPP 0.5mg/mL
n.d. Not determined
n.s. Not scorable due to strong cytotoxicity and/or poor metaphase quality
n.t. Not tested
EXPERIMENTAL RESULT
The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Experiment I without metabolic activation had to be terminated prematurely due to irregular cell growth at all concentrations. This part was repeated with modified concentrations in the absence of metabolic activation. The experimental part of the second experiment without metabolic activation was repeated as experiment IIA since the recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 was not covered.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor did not reach the threshold of three times the mutation frequency of the corresponding solvent controls in any of the experimental parts. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation and in the first culture of experiment IIA without metabolic activation. These increases however, were judged as irrelevant since the induction factor did not reach the threshold discussed above.
Summary of results:
| concentration (µg/ml) | S9 Mix | relative cloning efficiency I (%) | relative cloning efficiency II (%) | mutant colonies / 106 cells | induction factor | relative cloning efficiency I (%) | relative cloning efficiency II (%) | mutant colonies / 106 cells | induction factor | |
| Experiment I / 4h treatment | culture I | culture II | ||||||||
| solvent control (THF) | - | 100.0 | 100.0 | 11.3 | 1.0 | 100.0 | 100.0 | 15.0 | 1.0 | |
| positive control (EMS) | 150.0 | - | 69.4 | 90.5 | 90.8 | 8.0 | 71.7 | 98.8 | 95.9 | 6.4 |
| test item | 0.6 | - | 102.3 | culture was not continued# | 94.9 | culture was not continued# | ||||
| test item | 1.3 | - | 87.0 | 91.2 | 21.3 | 1.9 | 85.9 | 104.2 | 20.0 | 1.3 |
| test item | 2.5 | - | 86.2 | 90.3 | 10.1 | 0.9 | 87.7 | 87.7 | 12.5 | 0.8 |
| test item | 5.0 | - | 66.5 | 95.4 | 3.4 | 0.3 | 83.6 | 99.1 | 9.4 | 0.6 |
| test item | 10.0 | - | 57.1 | 94.7 | 10.6 | 0.9 | 72.5 | 95.8 | 24.4 | 1.6 |
| test item | 20.0 | - | 4.5 | 91.0 | 10.3 | 0.9 | 39.1 | 78.9 | 22.0 | 1.5 |
| test item | 40.0 | - | 0.0 | culture was not continued## | 3.4 | 95.3 | 32.4 | 2.2 | ||
| solvent control (THF) | + | 100.0 | 100.0 | 19.7 | 1.0 | 100.0 | 100.0 | 16.1 | 1.0 | |
| positive control (DMBA) | 1.1 | + | 55.4 | 71.6 | 1430.0 | 72.6 | 52.1 | 84.7 | 1109.9 | 69.0 |
| test item | 20.0 | + | 105.9 | 85.0 | 30.4 | 1.5 | 103.8 | 94.9 | 18.6 | 1.2 |
| test item | 40.0 | + | 96.4 | 99.0 | 17.7 | 0.9 | 98.6 | 88.1 | 18.4 | 1.1 |
| test item | 80.0 | + | 93.8 | 91.5 | 16.2 | 0.8 | 91.1 | 91.4 | 40.0 | 2.5 |
| test item | 160.0 | + | 15.1 | 86.8 | 3.9 | 0.2 | 88.5 | 86.8 | 38.8 | 2.4 |
| test item | 240.0 | + | culture was not continued## | 19.9 | 87.2 | 21.0 | 1.3 | |||
| 320.0 | + | culture was not continued## | culture was not continued## | |||||||
| Experiment II / 24h treatment | culture I | culture II | ||||||||
| solvent control (THF) | - | 100.0 | 100.0 | 17.6 | 1.0 | 100.0 | 100.0 | 21.9 | 1.0 | |
| positive control (EMS) | 150.0 | - | 82.9 | 54.6 | 681.5 | 38.7 | 90.2 | 58.1 | 606.2 | 27.7 |
| test item | 1.3 | - | 82.1 | culture was not continued# | 110.5 | culture was not continued# | ||||
| test item | 2.5 | - | 89.6 | culture was not continued# | 103.1 | culture was not continued# | ||||
| test item | 5.0 | - | 88.8 | 99.7 | 30.9 | 1.8 | 109.7 | 95.4 | 28.9 | 1.3 |
| test item | 10.0 | - | 95.0 | 96.8 | 16.7 | 1.0 | 110.9 | 93.8 | 19.2 | 0.9 |
| test item | 20.0 | - | 92.8 | 96.4 | 13.8 | 0.8 | 110.7 | 93.8 | 15.8 | 0.7 |
| test item | 40.0 | - | 90.1 | 87.1 | 23.4 | 1.3 | 111.1 | 93.2 | 15.4 | 0.7 |
| test item | 80.0 | - | 40.2 | 62.4 | 34.7 | 2.0 | 90.1 | 63.1 | 22.3 | 1.0 |
| Experiment II / 4h treatment | culture I | culture II | ||||||||
| solvent control (THF) | + | 100.0 | 100.0 | 15.8 | 1.0 | 100.0 | 100.0 | 30.1 | 1.0 | |
| positive control (DMBA) | 1.1 | + | 73.8 | 51.7 | 861.0 | 54.4 | 78.4 | 40.3 | 1067.7 | 35.5 |
| test item | 20.0 | + | 92.2 | culture was not continued# | 101.8 | culture was not continued# | ||||
| test item | 40.0 | + | 94.7 | 90.0 | 13.5 | 0.9 | 100.5 | 76.4 | 34.2 | 1.1 |
| test item | 80.0 | + | 94.0 | 100.8 | 11.1 | 0.7 | 89.7 | 96.9 | 21.5 | 0.7 |
| test item | 120.0 | + | 84.6 | 93.6 | 21.8 | 1.4 | 87.3 | 92.6 | 19.9 | 0.7 |
| test item | 180.0 | + | 50.8 | 95.8 | 17.6 | 1.1 | 82.0 | 80.5 | 32.1 | 1.1 |
| test item | 240.0 | + | 0.9 | 88.0 | 13.3 | 0.8 | 46.8 | 82.4 | 31.2 | 1.0 |
| Experiment IIA / 24h treatment | culture I | culture II | ||||||||
| solvent control (THF) | - | 100.0 | 100.0 | 18.4 | 1.0 | 100.0 | 100.0 | 35.1 | 1.0 | |
| positive control (EMS) | 150.0 | - | 93.0 | 87.5 | 374.9 | 20.4 | 95.2 | 109.6 | 313.1 | 8.9 |
| test item | 10.0 | - | 98.6 | 85.7 | 17.7 | 1.0 | 97.7 | 102.6 | 23.6 | 0.7 |
| test item | 20.0 | - | 95.7 | 87.4 | 20.6 | 1.1 | 95.6 | 99.5 | 34.6 | 1.0 |
| test item | 40.0 | - | 72.1 | 77.5 | 34.6 | 1.9 | 99.5 | 108.0 | 46.1 | 1.3 |
| test item | 80.0 | - | 8.9 | 82.0 | 44.5 | 2.4 | 56.8 | 88.6 | 44.6 | 1.3 |
| test item | 120.0 | - | 0.0 | culture was not continued## | 2.7 | culture was not continued## | ||||
| test item | 160.0 | - | 0.0 | culture was not continued## | 0.0 | culture was not continued## | ||||
# culture was not continued since a minimum of only four analysable concentrations is required
## culture was not continued due to exceedingly severe cytotoxic effects
cloning efficiency I (survival): cloning efficiency determined immediately after treatment to measure toxicity.
cloning efficiency II (viability): cloning efficiency determined after the expression period to measure viability of the cells without selective agent.
DETAILS ON EXPERIMENTAL RESULTS:
In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test article revealed no marked deviations.
| TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1538 | WP2uvrA | |||||||
| Dose (µg/0.1 ml) | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
| solvent control | 27 | 53 | 166 | 106 | 12 | 7 | 4 | 10 | 9 | 31 | 24 | 25 |
| 5 | 14 | 39 | 155 | 109 | 8 | 10 | 6 | 12 | 9 | 30 | 23 | 21 |
| 10 | 18 | 59 | 154 | 99 | 10 | 8 | 4 | 12 | 11 | 31 | 22 | 12 |
| 50 | 16 | 50 | 157 | 102 | 11 | 10 | 5 | 13 | 13 | 30 | 21 | 21 |
| 100 | 21 | 42 | 146 | 93 | 11 | 11 | 5 | 5 | 9 | 37 | 23 | 19 |
| 500 | 19 | 50 | 137 | 90 | 8 | 12 | 3 | 12 | 5 | 38 | 17 | 17 |
| 1000 | 15 | 55 | 171 | 101 | 10 | 9 | 2 | 13 | 10 | 27 | 16 | 17 |
| 5000 | 15 | 65 | 142 | 94 | 5 | 10 | 0 | 7 | 5 | 28 | 17 | 11 |
| positive controls: | ||||||||||||
| solvent control | 17 | 45 | 180 | 146 | 11 | 12 | 11 | 17 | 9 | 32 | 19 | 15 |
| concentration A | 385 | 1163 | 712 | 1131 | >1500 | 530 | 136 | 73 | ~1100 | 374 | 318 | 617 |
| concentration B | 533 | 1261 | 1187 | 1932 | >2000 | 1000 | 52 | >1500 | 280 | 430 | 764 | |
For details on positive controls see table 1 above.
EXPERIMENTAL RESULT Both in the presence and absence of metabolic activation, the test substance did not induce a statistically relevant increase in the number of chromosome aberrations.
Exposure/fixation/+/-S9 |
|||||
3h/24h/-S9 |
Ethanol (1.0% v/v) |
300 µg/ml |
400 µg/ml |
550 µg/ml |
MMC-C 0.5 µg/ml |
Mitotic Index (%) |
100 |
97 |
67 |
54 |
99 |
No. of cells with aberration (-gaps) |
2/200 |
1/200 |
1/200 |
1/200 |
51**/100 |
3h/24h/+S9 |
Ethanol (1.0% v/v) |
200 µg/ml |
300 µg/ml |
500 µg/ml |
CP 10 µg/ml |
Mitotic Index (%) |
100 |
98 |
85 |
41 |
58 |
No. of cells with aberration (-gaps) |
1/200 |
1/200 |
1/200 |
2/200 |
60**/100 |
24h/24h/-S9 |
Ethanol (1.0% v/v) |
200 µg/ml |
300 µg/ml |
350 µg/ml |
MMC-C 0.2 µg/ml |
Mitotic Index (%) |
100 |
103 |
69 |
43 |
54 |
No. of cells with aberration (-gaps) |
3/200 |
3/200 |
3/200 |
4/200 |
53**/100 |
48h/48h/-S9 |
Ethanol (1.0% v/v) |
100 µg/ml |
350 µg/ml |
400 µg/ml |
MMC-C 0.1 µg/ml |
Mitotic Index (%) |
100 |
77 |
58 |
48 |
81 |
No. of cells with aberration (-gaps) |
5/200 |
2/200 |
3/200 |
2/200 |
59**/150 |
3h/48h/+S9 |
Ethanol (1.0% v/v) |
400 µg/ml |
500 µg/ml |
750 µg/ml |
CP 10 µg/ml |
Mitotic Index (%) |
100 |
95 |
80 |
44 |
-* |
No. of cells with aberration (-gaps) |
1/200 |
7/200 |
2/200 |
2/200 |
48**/150 |
*CP was fixed after 24h; therefore, the mitotic index could not be calculated as percentage of control |
|||||
** significant (P 0.001) |
|||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The reigstered substance was negative in an in vivo micronucleus assay.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- January 15, 2013 - February 15, 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: pre-experiment: 8 – 9 weeks, main experiment: 9 - 10 weeks
- Weight at study initiation: mean value 36.7 g
- Assigned to test groups randomly: yes
- Housing: single in Makrolon Type II/III cages with wire mesh top(EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: pelleted standard diet, ad libitum, (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 45 - 65 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
- Amount of vehicle: 10 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in corn oil. - Duration of treatment / exposure:
- single treatment
- Frequency of treatment:
- once
- Post exposure period:
- 24 h preparation interval: 500, 1000, and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw - Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Seven males were assigned to each test group (except the vehicle and positive control groups with five animals each).
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- CPA; cyclophosphamide
- Route of administration: orally, once
- Volume administered: 10 mL/kg bw
- Doses / concentrations: 40 mg/kg bw - Tissues and cell types examined:
- Polychromatic erythrocytes (PCE) in the bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 0-1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were administered (500, 1000 and 2000 mg/kg), and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. Since no gender specific differences were observed in the pre-experiment, the main study was performed using males only in accordance with the test guidelines.
DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
OTHER:
The animals of all dose groups, except the positive control were examined for clinical signs at intervals of around 0-1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles. - Evaluation criteria:
- A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system. - Statistics:
- nonparametric Mann-Whitney test
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, Eyelid closure, Ruffled fur, Tumbling
RESULTS OF DEFINITIVE STUDY
In the main experiment clinical signs after treatment with the test item comprised ruffled fur (4/7 mice at 1000 mg/kg bw, 14/14 at 2000 mg/kg bw), reduction of spontaneous activity (3/7 mice at 1000 mg/kg bw and 11/14 at 2000 mg/kg bw) as well as tumbling (5/14 mice at 2000 mg/kg bw) No clinical symptoms were observed at 500 mg/kg bw.
After treatment with the test item at the 24 h and 48h preparation interval the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test article did not induce cytotoxic effects in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment were below to the value of the vehicle control group and well within the historical control data range. The positive control showed a statistically significant increase of induced micronucleus frequency. - Conclusions:
- Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse and is therefore considered to be non-mutagenic in this micronucleus assay.
- Executive summary:
A GLP-compliant study following OECD guideline 474 was performed to investigate the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated, based on results of a pre-experiment:
24 h preparation interval: 500, 1000, and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw
In the main experiment clinical signs after treatment with the test item comprised ruffled fur (4/7 mice at 1000 mg/kg bw, 14/14 at 2000 mg/kg bw), reduction of spontaneous activity (3/7 mice at 1000 mg/kg bw and 11/14 at 2000 mg/kg bw) as well as tumbling (5/14 mice at 2000 mg/kg bw) No clinical symptoms were observed at 500 mg/kg bw. After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test substance did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. The mean values of micronuclei observed after treatment were below to the value of the vehicle control group and well within the historical control data range. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test article is considered to be non-mutagenic in this micronucleus assay.
Reference
Summary of Micronucleus Test Results
| Test group | Dose(mg/kg bw) | Sampling time (h) | PCEs with micronuclei (%) | Range | PCE per 2000 erythrocytes |
| Vehicle control | 0 | 24 | 0.24 | 2 - 9 | 1212 |
| Test item | 500 | 24 | 0.143 | 1 - 5 | 1286 |
| Test item | 1000 | 24 | 0.121 | 0 - 8 | 1243 |
| Test item | 2000 | 24 | 0.214 | 1 - 9 | 1210 |
| Positive control | 40 | 24 | 2.7 | 33 - 86 | 1200 |
| Vehicle control | 0 | 48 | 0.15 | 2 - 4 | 1212 |
| Test item | 2000 | 48 | 0.114 | 0 - 5 | 1202 |
Biometry
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
| Vehicle control versus test group | Significance | p |
| 500 mg/kg bw; 24 h | n.t. | - |
| 1000 mg/kg bw; 24 h | n.t. | - |
| 2000 mg/kg bw; 24 h | n.t. | - |
| 40 mg CPA/kg bw; 24 h | + | 0.004 |
| 2000 mg/kg bw; 48 h | n.t. | - |
+ = significant;
n.t = not tested, as the mean micronucleus frequency was not above the vehicle control value
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Both the target and the source chemical have been investigated in several genotoxicity studies. The following data are available with the registered target chemical:
Ames test
The test article was tested for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophan-auxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml. In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of test material revealed no marked deviations. No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.
Similar results are reported in a supporting study, which showed no marked deviations in the comparison of the number of back-mutant colonies in the controls and the treated cultures either with or without microsomal activation.
in vitro chromosome aberration test
According to the results of the included in vitro cytogenetic study, the test substance led to a statistically significant and biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps both either without S9 mix or after the addition of a metabolizing system in a single experiment performed under light protection conditions with 4 hours treatment interval and 18 hours sampling time. In detail, in the 2nd Experiment after 4 hours treatment in the presence of metabolic activation a statistically significant increased chromosome aberration rate clearly exceeding the historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps) was obtained at 1250 μg/mL (18% aberrant metaphases excl. gaps) when handling the test substance without light exclusion. This finding was assumed as artificial observation due to missing light protection. However, in the 3rd Experiment, performed following the sponsors recent instructions on handling under light exclusion, the chromosome damaging effect was corroborated. The structural chromosome aberration rates of the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that the test substance is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.
In vivo micronucleus test
A GLP-compliant study following OECD guideline 474 was performed to investigate the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (Harlan, 2013). The test item was dissolved in corn oil and administered at the following dose levels: 500 and 1000 mg/kg (24h preparation interval) and 2000 mg/kg bw (24 and 48 h preparation interval). Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test substance did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. The positive control showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test article is considered to be non-mutagenic in this micronucleus assay.
The following data are available for the source chemical:
Ames test
The source chemical's potential to induce point mutations in bacteria was analyzed in an Ames test using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. After exposure to the test item in the concentrations of 675 and 2025 µg/0.1 ml without microsomal activation, the number of colonies of histidine-prototrophic mutants was reduced as a result of the inhibitory effects of the substance on the growth of the bacteria.
Mammalian gene mutation study
The source chemical was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster following OECD guideline 476. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. A third experiment without metabolic activation was performed since the recommended cytotoxic range of approximately 10-20% was not covered in the second experiment without metabolic activation. The third experiment, designated experiment IIA, was performed with a treatment time of 24 hours. The cell cultures were evaluated at the following concentrations: Experiment I: without S9 mix: 1.3; 2.5; 5.0; 10.0; and 20.0 μg/mL; with S9 mix: 20.0; 40.0; 80.0; and 160.0 μg/mL Experiment II: without S9 mix: 5.0; 10.0; 20.0; 40.0; and 80.0 μg/mL; with S9 mix: 40.0; 80.0; 120.0; 180.0; and 240.0 μg/mL Experiment IIA: without S9 mix: 10.0; 20.0; 40.0; and 80.0 μg/mL. The highest concentration of the main experiments was limited by the cytotoxicity of the test item. Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50% occurred in the first experiment at 20.0 µg/mL and above without metabolic activation and at 160 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 80.0 µg/mL without and 240 µg/mL with metabolic activation. In the additional experiment IIA, cytotoxicity was noted at 80.0 µg/mL and above. No substantial and reproducible dose dependent increase of the mutation frequency was observed in any of the main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
Chromosome aberration study
The ability of the source substance to induce chromosome aberrations was
evaluated in cultured peripheral human lymphocytes either in the
presence or absence of a metabolic activation system (S9-mix) in two
independent experiments. The study procedures described in this report
were based on the most recent OECD and EEC guidelines and in accordance
with internal SOPs. The test article was soluble in ethanol at
concentrations of up to 333 mg/ml. Initially, a dose-range finding test
was performed in order to select the dose levels for the main tests. In
the first cytogenetic assay, the test article was tested up to 550 and
500 µg/ml for a 3 h exposure time with a 24 h fixation time in the
absence and presence of 1.8% (v/v) S9-fraction, respectively. The
inhibition of the mitotic index for these dose levels was 46% and 59%,
respectively. In the second cytogenetic assay, the test substance was
tested up to 350 µg/ml for a 24 h continuous exposure time with a 24 h
fixation time and up to 400 µg/ml for a 48 h continuous exposure time
with a 48 h fixation time in the absence of S9-mix. The inhibition of
the mitotic index for these dose levels was 57% and 52%, respectively.
In the presence of S9-mix the test item was tested up to 750 µg/ml for a
3 h exposure time with a 48 h fixation time. The inhibition of the
mitotic index was 56% and precipitation in the culture medium at this
dose level was observed. Positive control chemicals both produced a
statistically significant increase in the incidence of cells with
chromosome aberrations, indicating that the test conditions were
adequate and that the metabolic activation system (S9-mix) functioned
properly. The test item did not induce a statistically significant or
biologically relevant increase in the number of cells with chromosome
aberrations in the absence and presence of S9-mix, in either of the two
independently repeated experiments. No effects on the number of
polyploid cells and cells with endoreduplicated chromosomes were
observed both in the absence and presence of S9-mix. Therefore it can be
concluded that the test substance does not disturb mitotic processes and
cell cycle progression and does not induce numerical chromosome
aberrations under the experimental conditions described in this report.
Finally, it is concluded that this test is valid and that the test
chemical is not clastogenic in human lymphocytes under the conditions of
this chromosome aberration assay.
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.
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