Registration Dossier
Registration Dossier
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EC number: 239-473-5 | CAS number: 15454-75-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In Vitro Ames: Kato (2003)
Under the conditions of the study, it is concluded from the foregoing results that the mutagenic activity of the test substance is considered negative. Neither toxic effect nor precipitation of the test substance was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation.
In Vitro Chromosome Aberration: Yokota (2003)
It was concluded that the test material induces chromosome aberrations in the cell line of Chinese hamster fibroblast in lung (CHL/IU cells). However, further review suggests genotoxicity observed with zinc is secondary to effects on other cellular components, and not due to direct interaction of zinc with DNA, or oxidative stress.
In Vitro Chromosome Aberration: Hargitai (2013) Read-Across
Under the conditions of the study, the test material did not induce reproducible, statistically significant increases in chromosome aberrations in either assay, with or without metabolic activation. Therefore, the test material is considered not clastogenic in this test system.
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:
- 29 August 2012 to 16 December 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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: Dulbecco’s Modified Eagle’s Medium.
- Properly maintained: Yes, stocks were kept in a freezer at -80 ± 10 °C. The laboratory cultures were maintained in 150 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air.
- Periodically checked for Mycoplasma contamination: Yes, no infection was noted. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Assay 1 and 2 test concentrations: 5000, 2500, 1250, 625 and 312.5 μg/mL (with and without metabolic activation).
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water.
- Justification for choice of solvent/vehicle: Based on the results of a short preliminary solubility test, the test material was soluble in distilled water at 500 mg/mL. Therefore, distilled water was selected as solvent for the study. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- Positive control solutions were prepared immediately before the treatment of the cells and filtered sterile using a 0.22 μm syringe filter before use.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium.
Cells were treated with test material solutions, negative (solvent) or positive control solution (treatment volume: 100 μL/dish in all cases) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with Dulbecco’s Modified Eagle’s Medium (supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
DURATION
- Exposure duration:
> Assay 1: 3 hours with and without S9-mix.
> Assay 2: 20 hours without S9-mix and 3 hours with S9-mix.
- Expression time:
> Assay 1: Cells were harvested 20 hours after the beginning of treatment (approximately 1.5 normal cell cycles).
> Assay 2: Cells were harvested 28 hours after the beginning of treatment (approximately 2 normal cell cycles).
SPINDLE INHIBITOR: Colchicine (0.2 μg/mL) was added to cultures 2 to 2.5 hours prior to harvesting.
The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol:Acetic acid 3:1 (v:v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried.
STAIN: The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.
NUMBER OF REPLICATIONS: Two replications were performed.
NUMBER OF CELLS EVALUATED: At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) had been recorded for that culture. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
DETERMINATION OF CYTOTOXICITY
- Method: For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.
OTHER EXAMINATIONS:
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. - Evaluation criteria:
- The assay is considered valid, if the following criteria are met:
- The solvent control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
A test material is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
A test material is concluded to have given a negative response if no reproducible, statistically significant increases are observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No large changes in the pH were observed.
- Effects of osmolality: A slightly higher than usual difference was observed in osmolality was observed at the highest concentration (5000 μg/mL) in both experiments of Assays 1 and 2.
- Water solubility: No insolubility was detected at the end of the treatment period in the final treatment medium for either Assay 1 or 2.
RANGE-FINDING/SCREENING STUDIES: A total of eight test concentrations between 5000 and 2.29 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
Cells survival relative to the negative solvent control ranged from (exposure time/harvest time):
> Assay A: 88 - 114% (3/20 without S9-mix) and 84 – 123 % (3/20 with S9-mix).
> Assay B: 77 - 127% (20/28 without S9-mix) and 88 – 116 % (3/28 with S9-mix).
All observations were normal throughout the study, pH measured 7.4 in all samples and osmolarity ranged from 321 to 402 mmol/kg.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative (solvent) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in both experiment in Assays 1 and 2.
CHROMOSOME ADERRATION EVALUATION
Concentrations of 5000, 2500 and 1250 μg/mL (a total of three) were chosen for evaluation in both cases in Assays 1 and 2. Based on the observed results, an additional concentration (625 μg/mL) was evaluated in Assay 1 in the experiment with metabolic activation.
None of the treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay without metabolic activation or in Assay 2 with metabolic activation. Following the initial analysis of three concentrations, a significant increase in chromosome aberrations at 1250 μg/mL in Assay 1 with metabolic activation was observed. No increase was found at two higher concentrations (2500 and 5000 μg/mL). A further concentration, 625 μg/mL, was then analysed and no increase in aberrations was observed. Therefore the increase was not repeatable either at other concentrations within the assay or between assays.
POLYPLOID AND ENDOREDUPLICATED METAPHASES
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-3) were found in some cases in the negative (solvent) control or test material treated samples in the performed experiments. No endoreduplicated metaphases were found in the main tests. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Under the conditions of the study, the test material did not induce reproducible, statistically significant increases in chromosome aberrations in either assay, with or without metabolic activation. Therefore, the test material is considered not clastogenic in this test system.
- Executive summary:
The clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with the standardised guideline OECD 473, EU Method B.10 and EPA OPPTS 870.5375.
Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. Both experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period, or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.
Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication of polyploidy or endoreplication.
The test material is therefore considered not to be clastogenic in this test system.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 April 2003 to 27 May 2003
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- other: Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No. 77 of JMOL on September 1, 1988) and Notification on partial revision of the standards (Notification No. 67 of JMOL on June 2, 1997).
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- other: Notification on partial revision of testing methods relating to new chemical substances (Notification No. 287 of Planning and Coordination Bureau, JEA, No. 127 of Pharmaceutical Affairs Bureau, JMHW, No. 2 of Basic Industries Bureau, JMITI 31/10/1997
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium: Histidine
Escherichia coli: Tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: The bacterial strains were chosen for the reason to take account of their high sensitivity to known mutagens, and these bacterial strains were the commonest tester strain to be used in this sort of the test.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The liquid medium of Nutrient broth No.2 (OXOID) was used for preparation of bacterial suspensions. The minimum glucose agar plates used in this test were purchased from Kyokuto Pharmaceutical Industrial Co., Ltd. (Japan) and stored in the room temperature until use. The minimum glucose agar plates (prepared on January 31, 2003) were prepared in accordance with the guideline of the reverse mutation test using Microorganisms (JMHLW).
The overlay agar used in the test of Salmonella typhimurium TA strains consisted of 10 part of the agar solution which include 0.6 % of agar (Bacio-Agar, Difeo) and 0.5 % of NaCl and of 1 part of the 0.5 mM D-biotin and L-histidine solution. - Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: The bacterial strains were chosen for the reason to take account of their high sensitivity to known mutagens, and these bacterial strains were the commonest tester strain to be used in this sort of the test.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The liquid medium of Nutrient broth No.2 (OXOID) was used for preparation of bacterial suspension. The minimum glucose agar plates used in this test were purchased from Kyokuto Pharmaceutical Industrial Co., Ltd. (Japan) and stored in the room temperature until use. The minimum glucose agar plates (prepared on January 31, 2003) were prepared in accordance with the guideline of the reverse mutation test using Microorganisms (JMHLW).
The overlay agar used in the test of Escherichia coli WP2 uvrA included 0.5 mM L-tryptophan solution. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary test: 5, 20, 78, 313, 1 250, 5 000 µg/plate
Main test: 313, 625, 1 250, 2 500, 5 000 µg/plate
The preliminary test was performed to determine the most favourable dose levels of the test substance in the main test. The maximum dose of the preliminary test was set at 5 000 μg/plate in accordance with the test guideline applied, and total six different dose levels with factor of 4 from the maximum dose were employed.
Neither toxic effect of the test substance nor two-fold or more increase in the number of the revertant colonies with dose-relativity compared with the negative control was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation. Moreover, precipitation of the test substance was not detected in any dose levels. From the foregoing results, the maximum dose of the main test of all bacterial strains in both the presence and the absence of metabolic activation was set at 5 000 μg/plate and total five different dose levels with factor of 2 from the maximum dose were employed. - Vehicle / solvent:
- - Vehicle/ solvent used: Distilled water
- Justification for choice of solvent/vehicle: Distilled water was used as the solvent of the test substance by the reason that the test substance was stable in water, and that the solubility in water of the test substance was 10 wt% or more. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- benzo(a)pyrene
- furylfuramide
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Pre-incubation
All bacterial strains obtained were suspended in Nutrient broth No.2 (OXOID) and were stored at -80 °C. Before the examination, the bacterial strains were re-isolated from the frozen suspension by streaking it on a Nutrient broth agar plate and incubated at 37 'C. The grown reisolated colonies were then resuspended in a Nutrient broth No.2 (OXOID) and incubated at 37 °C with shaking until these bacterial strains were attained to the early stationary phase.
After incubation, appropriate volume of dehydrated spectrophotometric grade dimethylsulfoxide was added to each bacterial suspension at the ratio of 0.07 mL to 0.8 mL and stored at -80 °C until use. The genetic markers, such as auxotrophic mutation (histidine or tryptophan requirement), rfa mutation, uvrA or uvrB mutation, number of spontaneous revertants, R-factor, sensitivity of these bacterial strains to known mutagens were checked in advance before using the bacterial strains. And it was confirmed that the genetic markers and sensitivity of these bacterial strains were suitable.
NUMBER OF REPLICATIONS: Test material, vehicle and positive controls were plated in duplicate.
METHOD OF APPLICATION: In agar (plate incorporation)
An amount of 0.1mL of the test substance solution was put in the test tube. In the test of the absence of metabolic activation, 0.5 mL of 0.1 M phosphate buffer (pH 7.4) and 0.1 mL of the bacterial suspension were added to the test substance solution and then incubated at 37 °C for 20 min with shaking. In the test of the presence of metabolic activation, 0.5 mL of 0.1 M phosphate buffer was replaced by 0.5 mL of S9 Mix. The suspension was then thoroughly mixed with 2.0 mL of the overlay agar and the mixture was poured over the surface of the minimum glucose agar plate. The number of the minimum glucose agar plate of each dose level was used in duplicate. Both the negative and the positive controls were similarly carried out. Sterility test of S9 Mix, 0.1 M phosphate buffer, Nutrient broth No.2 and the test substance solution of the maximum concentration were performed to confirm the existence of the contaminant. After the incubation at 37 °C for 48 hours, number of revertant colonies per plate were counted. The measuring of the number of the revertant colonies was carried out using colony analyser that was set up to correct the measuring value. Toxic effect of the test substance was examined with a stereoscopic microscope and precipitation of the test substance was observed by naked eyes. The individual number of the revertant colonies and their mean value were expressed as the results of each dose level.
DURATION: 48 hours
- PREPARATIION OF BACTERIAL SUSPENSION
The frozen bacterial suspension was thawed, 100 μL of the thawed bacterial suspension was inoculated in 30 mL of Nutrient broth No.2 (OXOID) and stored in a refrigerator until the time of the cultivation start. The rest-thawed suspension used for inoculation was not reused. The bacterial suspension was then incubated at 31 °C for 10 hours with shaking. The optical density of this growing culture was measured with the photoelectric spectrophotometer (630 nm), and it was confirmed that it was approximately 1 x 10^9 cells per mL or more by converting the value of the optical density.
PREPARATION OF THE TEST SUBSTANCE SOLUTION
The test substance of predetermined quantity was weighed when used. The highest concentration of the test substance solution used in this test was prepared using distilled water just before use. The solutions of the lower concentration were prepared by the same distilled water by serial dilution from the solution of the highest concentration. The weight value of the test substance was not converted into the purity in the time of the preparation.
PREPARATION OF CONTROLS
Preparation of positive controls: NaN3 was dissolved in distilled water (Otsuka Pharmaceutical Factory, Inc.) and other positive controls were dissolved in dimethylsulfoxide (Wako Pure Chemical Industries, Ltd.). These positive control solutions were diluted in the used concentration and stored at -20 °C until use. The retention period of the positive control solution was less than 1 year, and the positive control solution that was diluted in the concentration to use was less than 3 months, respectively. It is confirmed that the positive controls stored are not declining the mutagenic activity for 1 year or more. - Evaluation criteria:
- The number of the revertant colonies of the negative control and positive control in all bacterial strains was compared with the values of the historical data in our laboratory. The values of the negative control and the positive control were judged to satisfy acceptance criteria when either of the next two criteria are filled. One of the criteria is that the number of the revertant colonies is within the range of the standard value (mean ± 2 SD) of the historical data. Another criterion is although the number of the revertant colonies is out of the range in the standard value, it is judged to be appropriate value because it occurs by chance from the comparison with the historical data.
When the experiment satisfies the following criteria, it was judged that the test result was obtained from the test of the appropriate procedure.
1) The growth of the contaminant in the test materials, such as Nutrient broth, the test substance solution of the highest concentration, S9 Mix, sodium phosphate buffer are not detected from the result of the sterility test.
2) The negative control values satisfy acceptance criteria.
3) The positive control values satisfy acceptance criteria. And the number of the revertant colonies of the positive control increased two-fold or more compared with the negative control.
Judgement of the test results
When the negative control and the positive control values satisfy acceptance criteria and the test results satisfy acceptance criteria, the test substance was judged positive for mutagenic activity when clear dose-related increase in the number of the revertant colonies and two-fold or more increase in the number of the revertant colonies compared with the negative control were observed with reapperance. - Statistics:
- No statistical analysis was used for evaluation of the test results.
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Neither clear dose-related increase in the number of the revertant colonies nor two-fold or more increase in the number of the revertant colonies compared with the negative control was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation. And the reapperance of the test results between the preliminary and the main test was confirmed. On the other hand, the growth of the contaminant was not observed in a result of the sterility test. The numbers of the revertant colonies of the negative control and the positive control were within the range of the standard value of the historical data in our laboratory. Furthermore, all of 2-(2-furyl)-3-(5-nitro-2- furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[a ]pyrene and 2-aminoanthracene used as the positive controls increased the number of the revertant colonies two-fold or more compared with the negative control with all bacterial strains, respectively. These results indicate that the test has been properly carried out.
- Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: Negative with and without metabolic activation
Under the conditions of the study, it is concluded from the foregoing results that the mutagenic activity of the test substance is considered negative. Neither toxic effect nor precipitation of the test substance was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation. - Executive summary:
The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted under GLP conditions. In this study, the test material was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in duplicate.
Neither toxic effect of the test material nor two-fold or more increase in the number of the revertant colonies with dose-relativity compared with the negative control was observed in any bacterial strains at any dose level regardless of the presence or the absence of metabolic activation. Moreover, precipitation of the test material was not detected in any dose levels.
Under the conditions of this study, it is concluded from the foregoing results that the mutagenic activity of the test substance is considered negative in both the presence and absence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 October 2003 to 23 January 2004
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- “Standard for investigation by Chromosome Aberration Test using Cultured Mammalian Cells and revision of the form for Report of results of Chromosomal Aberration Test using cultured mammalian Cells” (Labour Standards Bureau Notification, kihatsu No. 652 September 29, 1997).
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- "Testing Methods for New chemical Substances" July 13, 1974 (Kampogyo No. 5, Yakuhatsu No. 615, and 49 Kikyoku No. 392), The director-general for the Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare, the director-general for the Manufacturing Industries Bureau, the Ministry of Economy, Trade and Industry and the director-general for the Environmental Policy Bureau, the Ministry of the Environment joint signature notification, revised on December 5, 1986 (Kampogyo No. 700, Yakuhatsu No. 1039, and 61 Kikyoku No. 1014), revised on March 31, 1987 ( Kampogyo No. 237, Yakuhatsu No. 306, 62 Kikyoku No. 303), revised on October 31, 1997 (Kampoan No. 287, Eisei No. 127 and Heisei 09.10.31 Kikyoku No.2) and revised on October 8, 1998 (Kampoan No. 443, Seieihatsu No. 1481 and Heisei 10.10.01 Kikyoku No.1).
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- “The Standard to be satisfied by the Test Facility etc. under the Provisions of Article 34-3(2) of the Ordinance on Industrial Safety and Health (Notice of the Ministry of Labor No.76, 1988) and amendments thereto.”
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- "Basic Standards to be observed by Testing Facilities conducting Tests stipulated in Article 4 of the Ordinance prescribing the Items of the Test relating to the New Chemical Substances and Study on Harmful Effects of the Designated Chemical Substances" March 31, 1984 (Kampogyo No. 39, Yakuhatsu No. 229, and 59 Kikyoku No. 85), The director-general for the Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare, the director-general for the Manufacturing Industries Bureau, the Ministry of Economy, Trade and Industry and the director-general for the Environmental Policy Bureau, the Ministry of the Environment joint signature notification, revised on November 18, 1988 (Kankiken No. 233, Eisei No. 38 and 63 Kikyoku No. 823) and revised on March 1, 2000 (Kampoan No. 41, Seieihatsu No. 268 and Heisei 12.02.14 Kikyoku No. 1).
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was dissolved in physiological saline and used for the experiments. The final concentration of physiological saline in the culture medium was less than 10 %. The maximum concentration of the test substance in the cell growth inhibition test was 3.580 mg/mL. Test solution for the maximum concentration was prepared, which was fully stirred with mixer after the test substance was weighed and the solvent was added. This test solution for the maximum concentration prepared was sterilized by filtration using membrane filter (0.22 µm diameter opening, MILLIPORE Corp.).
The test solutions were prepared just prior to testing.
The amount of test substance and the volume of solvent used for the preparation of test solution for the maximum concentration in each experiment were as follows. The test solutions in each experiment were prepared by diluting from the test solution for the maximum concentration to designated concentrations.
Cell growth inhibition test (prepared maximum concentration of test solution: 39.38 mg/mL).
The amount of test substance: 287.1 mg.
The volume of solvent (physiological saline): 7.291 mL.
Chromosome aberration test (prepared maximum concentration of test solution: 39.38 mg/mL).
The amount of test substance: 328.7 mg.
The volume of solvent (physiological saline): 8.347mL.
The prepared maximum concentration test solution for the chromosome aberration test was prepared by a dilution after it was made in the same concentration which was used in the cell growth inhibition test to avoid the preparation error between the cell growth inhibition test and the chromosome aberration test. - Species / strain / cell type:
- mammalian cell line, other: CHL/IU
- Details on mammalian cell type (if applicable):
- CELLS USED
- Cell cycle length, doubling time or proliferation index: The cells grow in a monolayer on a plate and the doubling time is approximately 15.8 hours. The cells were cultured every three to five days.
- Number of passages if applicable:
Cell growth inhibition test: 7 passages
Chromosome aberration test short-term exposure experiments: 9 passages
- Methods for maintenance in cell culture if applicable: Cells were stored in the deep freezer (-80 °C). 10 v/v% of Dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries, Ltd., purity: >99 %, lot No. LDL5944) was added to the culture medium at the time of storage.
- Modal number of chromosomes: The cells have a modal chromosome number of 25.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle’s MEM (GIBCO/ Lot No. 1158872) supplemented with 10 % calf serum (HyClone/ Lot No. AJA9537, inactivation for 30 minutes at 56 °C) was used as cell culture medium. The cell cultures were incubated using CO2 gas incubator (TE-HER, CP series CPD-172M: Hirasawa, INC.) under the conditions of a highly humidified atmosphere with 5 % carbon dioxide at 37 °C.
- Properly maintained: Yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Short-term exposure experiment without S9 Mix: 0.007, 0.014, 0.028, 0.056, 0.112, 0.224, 0.448, 0.895, 1.790 and 3.580 mg/mL
Short-term exposure experiment with S9 Mix: 0.007, 0.014, 0.028, 0.056, 0.112, 0.224, 0.448, 0.895, 1.790 and 3.580 mg/mL
Chromosome aberration test without S9 mix; 35, 45, 55 and 65 μg/mL
Chromosome aberration test with S9 mix; 25, 35, 45 and 55 μg/mL
The dose range in the chromosome aberration test was selected in accordance with the results of the cell growth inhibition test. In the results of the cell growth inhibition test, the concentrations of 50 % cell growth inhibition in short-term exposure experiments were 0.052 mg/mL (without S9 mix) and 0.045 mg/mL (with S9 mix). Based on the above results, the doses were selected for the chromosome aberration test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Physiological saline
- Justification for choice of solvent/vehicle: Since this test substance was water soluble from the information which was provided by the sponsor, the solubility tests in physiological saline were performed in our laboratory. In the solubility test, the test substance dissolved at 39.38 mg/mL in physiological saline, therefore, physiological saline was chosen as solvent for this study. Evolution of gas, exothermic reaction, etc. in physiological saline were not observed. - Untreated negative controls:
- yes
- Remarks:
- Physiological saline
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Physiological saline
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- mitomycin C
- Remarks:
- Without S9 Mix: Mitomycin C at 0.05 μg/mL. With S9 Mix: N-nitrosodimethylamine at 0.4 mg/mL The positive controls selected for the experiment are known clastogens and easily soluble in water.
- Details on test system and experimental conditions:
- CELL GROWTH INHIBITION TEST:
- Short-term Exposure Experiment without S9 mix: One dish per test dose was used. 2 x 10^4 cells in 5.0 mL of culture medium were seeded into a plastic dish (60 mm in diameter) and incubated for 3 days. The maximum concentration of test substance was 3.580 mg/mL, and the lower concentrations were diluted from the maximum concentration by a common ratio of 2. Ten applied concentrations (0.007, 0.014, 0.028, 0.056, 0.112, 0.224, 0.448, 0.895, 1.790 and 3.580 mg/mL) were set up. The solvent control group (physiological saline) was set up as negative control. 0.5 mL of each concentration test solution or solvent were added to the culture medium in dish and treated for 6 hours at 37 °C. The culture medium in the dish was not removed in the above process.
After the treatment, the culture medium in the dish was removed and the cells in the dish were washed with physiological saline. 5 mL of new culture medium were added and incubated for 18 hours (recovery). After the incubation, the culture medium in the dish was removed and the surface of the cells was washed with physiological saline. The cells were stained in 0.1 % crystal violet solution after fixation by 10 % formaldehyde.
A cell density was measured by using monolayer cultured cells densimeter, which classifies according to the shade of the cellular dye.
- Short-term Exposure Experiment with S9 mix: The cells were seeded in the same method as described in the short-term exposure experiment without S9 mix, and were incubated for 3 days. The dose levels, the number of dishes per dose level and negative control groups were in the same conditions as described in the short-term exposure experiment without S9 mix. 0.83 mL of S9 mix were added to the dish after 0.83 mL of culture medium were removed, which was set up as S9 mix diluted solution (final concentration of S9 was 5 %). Additionally, 0.5 mL of each test solution or solvent were added to the culture medium and treated for 6 hours at 37 °C. The culture medium in the dish was not removed in the above process. After the treatment, the same operations as described in the short-term exposure experiment without S9 mix were performed.
CHROMOSONE ABERRATION TEST:
- Short-term Exposure Experiment without S9 mix: 4 plastic dishes of 60 mm diameter (chromosome specimen: 2 dishes, cell growth rate: 2 dishes) per dose level were prepared. 2.0 x 10^4 cells in 5.0 mL of culture medium were seeded into a plastic dish (60 mm in diameter) and incubated for 3 days.
The maximum concentration of test substance was 65 μg/mL and lower concentrations were made by dilution from the maximum concentration solution by a common difference of 10 μg/mL. Four doses were examined.
Untreated control and solvent control were set up as negative control groups. Mitomycin C (0.05 μg/mL) was used for the positive control group. For the untreated control group, dishes were incubated as they were for 6 hours at 37 °C. In other dishes, 0.5 mL of the test solutions for each dose level or the solvent or the positive control solution were added. The culture medium in the dishes was not removed in the above process. These were treated for 6 hours at 37 °C. After the treatment, the culture medium in the dishes was removed and the cells were washed with physiological saline, and then 5 mL of new culture medium were added to the dishes. The cells were cultured for 18 more hours (recovery). After the culture, the specimen preparations for the chromosome aberration test were prepared using the dishes for chromosome specimen. The cell growth rates were measured using the dishes for cell growth rate.
To stop mitosis at metaphase, colcemid was added by 0.2 μg/mL as final concentration to dishes for the chromosome specimen 2 hours before the end of the culture.
After incubation with colcemid, the culture medium in each dish was transferred to a centrifuging tube (Spitz tube). 2 mL of 0.25 % trypsin were immediately added to the dish and the cells were detached from the dish, and then these were collected into the same centrifuge tube and centrifuged (1 000 rpm, 5 minutes). After the supernatant in the centrifuge tube was discarded, 5 mL of 0.075M KCl were added for hypotonic treatment and incubated for 15 minutes in water bath at 37 °C. The cells were semi-fixed by addition of 0.5 mL of cold fixative which was mixture of cold methanol and acetic acid (3:1). It was centrifuged immediately for 5 minutes at 1 000 rpm and 5 mL of fresh fixative were added. The cells were completely fixed after repeating the above operation 3 times. The cell suspension that became muddy lightly was prepared and dropped onto the clean slides, and then these slides were left to air-dry. The slides were dyed in 1.7 % Giemsa solution for 15 minutes.
The dishes for the measurement of cell growth rate were fixed and stained in the same way as described in the cell growth inhibition test, and then cell growth rates were measured.
- Short-term Exposure Experiment with S9 mix: The number of dishes per dose level was the same conditions as described in the short-term exposure experiment without S9 mix. The cells were seeded in the same methods as described in the short-term exposure experiment without S9 mix, and were incubated for 3 days.
The maximum concentration of test substance obtained from the results of cell growth inhibition test was 55 μg/mL, and lower concentrations were made by dilution from the maximum concentration solution by a common difference of 10 μg/mL. Four doses were examined.
Untreated control and solvent control were set up as the negative control groups. N-nitrosodimethylamine (0.4 mg/mL) was used for the positive control group. 0.83 mL of S9 mix were added to the dish after 0.83 mL of culture medium were removed, which was set up as the S9 mix diluted solution (final concentration of S9 was 5 %).
For the untreated control group, dishes were incubated as they were for 6 hours at 37 °C. In other dishes, 0.5 mL of the test solutions for each dose level or solvent or positive control solution were added. The culture medium in the dishes was not removed in the above process. These were treated for 6 hours at 37 °C.
After the treatment, the same operations as described in the short-term exposure experiment without S9 mix were performed.
- Preparation of Chromosome Specimen
Preparation interval (cell harvest) of each group and experimental designs were as follows.
Short-term exposure experiment without S9 mix:
Exposure period 6 hours
Recovery: 18 hours
Cell harvest: 24 hours
Short-term exposure experiment with S9 mix:
Exposure period 6 hours
Recovery: 18 hours
Cell harvest: 24 hours
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
200 consecutive well-spread metaphases (100 metaphases per dish) obtained from each dose group were observed under the microscopes. The number of cells carrying structural chromosome aberrations and their aberration type were recorded. The number of polyploid cells was determined at the same time (cells carrying greater than 37 chromosomes including triploid were recorded as polyploidy). These observations were performed under blind method for objectivity.
The type of chromosome aberration was classified as follows:
Gaps are the unstained part on the vertical axis of the chromatid, whose width shows less than the width of the chromatid and the form is clear.
Breaks are the unstained part on the vertical axis of the chromatid, whose width shows more than the width of the chromatid and the form is clear, or that the fragments deviate from the axis of the chromosome or chromatid.
Exchanges are defined as the mutual exchange induced by two or more cuttings of chromosomes or chromatid.
The other structure aberrations were defined as “others”. - Evaluation criteria:
- A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5 %
Equivocal (±) 5 % or more, less than 10 %
Positive (+) 10 % or more
When more than 50 cells in metaphase per dish was not observed by the cytotoxicity, it was excluded from the judgment, and recorded as “TOX”.
The short-term exposure experiments were performed first. When the result of the short-term exposure experiment was positive, the continuous exposure experiment wasn't to be performed. When the results of the short-term exposure experiment with and without S9 mix were negative, the continuous exposure experiment was to be performed. Furthermore, when the result of chromosome aberration test was positive but not definitely dose-dependent (the positive result was found only in a single dose group) or that was equivocal (±), a confirmatory test was to be performed including the same dose level which was positive. In the results of the confirmatory test, it was to be judged equivocal if the equivocal result was observed again, and to be judged positive if the positive result was induced without dose-dependence. - Statistics:
- No statistical analysis was performed to the results.
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the results of the chromosome aberration test both with and without S9 mix, the frequencies of cells carrying polyploidy were less than 5 % at any concentration. However, the frequencies of cells carrying structural chromosome aberrations without S9 mix increased to 28.5 and 15.5 % at the dose levels of 55 and 65 μg/mL, respectively. The results of that with S9 mix increased to 11.0 and 12.5 % at the dose levels of 45 and 55 μg/mL, respectively. Therefore these results were judged positive.
In the positive (MMC) control group in short-term exposure experiment without S9 mix, the frequency of cells carrying structural chromosome aberrations was 15.5 %. In the positive (DMN) control group in the short-term exposure experiment with S9 mix, the frequency of cells carrying structural chromosome aberrations was 40.0 %. These results showed that the experiments were performed appropriately.
The values of D20 which indicates structural aberrations of 20 % in the short-term exposure experiment both with and without S9 mix were 0.06 mg/mL.
The continuous exposure experiments were not performed because the results of the short-term exposure experiments were positive.
Based on the above results, it is concluded that this test substance induces chromosome aberrations in CHL/IU cells.
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The precipitation of the test substance in culture medium was observed at more than 1.790 mg/mL in the cell growth inhibition test. - Conclusions:
- Interpretation of results: Positive
Under the conditions of this study, it is concluded that the test material induces chromosome aberrations in the cell line of Chinese hamster fibroblast in lung (CHL/IU cells). - Executive summary:
The genotoxic potential of the test material was investigated in a study which was conducted under GLP conditions and following a method which was similar to that which is outlined in the standardised guideline OECD 473.
This in vitro chromosome aberration test in cultured mammalian cells (CHL/IU cells) was performed to assess the potential of the test material to induce chromosome aberrations.
The maximum concentration used in cell growth inhibition test was 3.580 mg/mL. Physiological saline was chosen as solvent for this study.
The precipitation of the test substance in culture medium was observed at more than 1.790 mg/mL in the cell growth inhibition test with and without a metabolic activation system (hereinafter referred to as “with S9 mix” and “without S9 mix”).
In the results of the cell growth inhibition test, the concentrations of 50 % cell growth inhibition in short-term exposure experiments without S9 mix and with S9 mix were 0.052 mg/mL and 0.045 mg/mL. Therefore, the maximum concentration of test substance without S9 mix was 65 μg/mL, and 4 applied concentrations which were prepared with dilution by a common difference of 10 μg/mL were set up and examined. In with S9 mix, the maximum concentration of test substance was 55 μg/mL, and 4 applied concentrations, prepared with dilution by a common difference of 10 μg/mL, were set up and examined.
In the results of the chromosome aberration test both with and without S9 mix, the frequencies of cells carrying polyploidy were less than 5 % at any concentration. However, the frequencies of cells carrying structural chromosome aberrations without S9 mix increased to 28.5 and 15.5 % at the dose levels of 55 and 65 μg/mL, respectively. The results of that with S9 mix increased to 11.0 and 12.5 % at the dose levels of 45 and 55 μg/mL, respectively. Therefore, the results were judged positive.
In the positive control group in each experiment, the frequencies of cells carrying structural chromosome aberrations were suitable values showing that the experiments were performed appropriately. As the results of the short-term exposure experiments were judged positive, the continuous exposure experiments were not performed.
Based on the above results, it is concluded that the test material induces chromosome aberrations in the cell line of Chinese hamster fibroblast in lung (CHL/IU cells).
Referenceopen allclose all
Table 1: Summary Table of Chromosome Aberration Assay 1
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
Relative Survival (%) # |
Mean % Aberrant Cells ## |
- |
Negative (solvent) Control |
3h/20h |
100 |
0.5 |
5000 |
3h/20h |
95 |
0.0 |
|
2500 |
3h/20h |
115 |
0.5 |
|
1250 |
3h/20h |
109 |
2.0 |
|
625 |
3h/20h |
83 |
NE |
|
312.5 |
3h/20h |
101 |
NE |
|
Positive Control |
3h/20h |
80 |
10.5*** |
|
+ |
Negative (solvent) Control |
3h/20h |
100 |
1.0 |
5000 |
3h/20h |
90 |
0.0 |
|
2500 |
3h/20h |
96 |
3.5 |
|
1250 |
3h/20h |
92 |
6.5** |
|
625 |
3h/20h |
102 |
3.0 |
|
312.5 |
3h/20h |
100 |
NE |
|
Positive Control |
3h/20h |
59 |
100.0*** |
Negative (solvent) control: 1% (v/v) Distilled water
Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
# compared to the negative (solvent) control
## excluding gaps
** p < 0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control.
*** p < 0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.
Table 2: Summary Table of Chromosome Aberration Assay 2
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
Relative Survival (%) # |
Mean % Aberrant Cells ## |
- |
Negative (solvent) Control |
20h/28h |
100 |
2.0 |
5000 |
20h/28h |
81 |
2.0 |
|
2500 |
20h/28h |
87 |
0.5 |
|
1250 |
20h/28h |
85 |
0.5 |
|
625 |
20h/28h |
95 |
NE |
|
312.5 |
20h/28h |
83 |
NE |
|
Positive Control |
20h/28h |
62 |
30.4*** |
|
+ |
Negative (solvent) Control |
3h/28h |
100 |
1.5 |
5000 |
3h/28h |
85 |
2.5 |
|
2500 |
3h/28h |
85 |
1.5 |
|
1250 |
3h/28h |
83 |
2.0 |
|
625 |
3h/28h |
93 |
NE |
|
312.5 |
3h/28h |
89 |
NE |
|
Positive Control |
3h/28h |
35 |
100.0*** |
Negative (solvent) control: 1% (v/v) Distilled water
Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
# compared to the negative (solvent) control
## excluding gaps
*** p < 0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.
Table 3: Summary Table of Polyploidy and Endoreduplicated Cells Assay 1
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
No. of Cells Observed |
|
Polyploid |
Endoreduplicated |
|||
- |
Negative (solvent) Control |
3h/20h |
0 |
0 |
1250 |
3h/20h |
2 |
0 |
|
2500 |
3h/20h |
0 |
0 |
|
5000 |
3h/20h |
3 |
0 |
|
Positive Control |
3h/20h |
0 |
0 |
|
+ |
Negative (solvent) Control |
3h/20h |
0 |
0 |
625 |
3h/20h |
0 |
0 |
|
1250 |
3h/20h |
1 |
0 |
|
2500 |
3h/20h |
2 |
0 |
|
5000 |
3h/20h |
1 |
0 |
|
Positive Control |
3h/20h |
0 |
0 |
|
Table 4: Summary Table of Polyploidy and Endoreduplicated Cells Assay 2
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
No. of Cells Observed |
|
Polyploid |
Endoreduplicated |
|||
- |
Negative (solvent) Control |
20h/28h |
1 |
0 |
1250 |
20h/28h |
0 |
0 |
|
2500 |
20h/28h |
2 |
0 |
|
5000 |
20h/28h |
0 |
0 |
|
Positive Control |
20h/28h |
0 |
0 |
|
+ |
Negative (solvent) Control |
3h/28h |
0 |
0 |
1250 |
3h/28h |
0 |
0 |
|
2500 |
3h/28h |
0 |
0 |
|
5000 |
3h/28h |
2 |
0 |
|
Positive Control |
3h/28h |
0 |
0 |
|
Table 1: Reverse mutation test of the test material using Salmonella typhimurium and Escherichia coli
Compound |
Dose (µg/plate) |
Mean number of revertant colonies |
||||
|
|
Base-pair substitution type |
Frameshift type |
|||
Mean number of revertant colonies in the absence of metabolic activation |
||||||
|
|
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
Distilled Water |
- |
134 |
8 |
30 |
31 |
10 |
Test material |
313 |
133 |
9 |
37 |
30 |
9 |
625 |
136 |
9 |
31 |
30 |
10 |
|
1250 |
137 |
7 |
26 |
28 |
10 |
|
2500 |
135 |
10 |
33 |
31 |
12 |
|
5000 |
133 |
9 |
41 |
27 |
10 |
|
AF-2 |
0.01 |
799 |
|
168 |
|
|
|
0.1 |
|
|
|
572 |
|
NaN₃ |
0.5 |
|
431 |
|
|
|
9AA |
80.0 |
|
|
|
|
624 |
Mean number of revertant colonies in the presence of metabolic activation |
||||||
Distilled water |
- |
142 |
10 |
31 |
37 |
12 |
Test material |
313 |
146 |
10 |
40 |
38 |
13 |
625 |
145 |
12 |
36 |
35 |
12 |
|
1250 |
141 |
11 |
31 |
41 |
13 |
|
2500 |
146 |
12 |
33 |
34 |
11 |
|
5000 |
148 |
11 |
34 |
34 |
11 |
|
B[a]P |
5.0 |
1247 |
|
|
351 |
65 |
2AA |
2.0 |
|
179 |
|
|
|
|
10.0 |
|
|
1015 |
|
|
Positive control concentrations:
Strain |
Absence of S9 Mix (µg/plate) |
Presence of S9 Mix (µg/plate) |
TA100 |
AF-2 (0.01) |
B[a]P (5.0) |
TA1535 |
NaN₃ (0.5) |
2AA (2.0) |
WP2 uvrA |
AF-2 (0.01) |
2AA (10.0) |
TA98 |
AF-2 (0.1) |
B[a]P (5.0) |
TA1537 |
9AA (80.0) |
B[a]P (5.0) |
Results of the Chromosome Aberration Test Without S9-Mix.
Concentration (µg/mL) |
Cells scored |
Cell growth rate (%) |
Number of aberrations (%) |
Number and percentage (%) of cells carrying structural aberrations |
Judgement |
|||||||
No. of polyploids |
Judgement |
g |
ctb |
cte |
csb |
cse |
TA |
TAG |
||||
Untreated control |
100 |
- |
0 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
200 |
- |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|||
Solvent control (physiological saline) |
100 |
100 |
0 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
200 |
(100) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|||
35 |
100 |
93 |
0 |
- |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
- |
100 |
94 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
200 |
(94) |
0 (0) |
0 (0) |
1 (0.5) |
0 (0) |
0 (0) |
0 (0) |
1 (0.5) |
1 (0.5) |
|||
45 |
100 |
87 |
1 |
- |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
- |
100 |
84 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
|||
200 |
(86) |
1 (0.5) |
0 (0) |
1 (0.5) |
1 (0.5) |
0 (0) |
0 (0) |
2 (1.0) |
2 (1.0) |
|||
55 |
100 |
60 |
3 |
- |
0 |
11 |
22 |
0 |
3 |
26 |
26 |
+ |
100 |
56 |
2 |
0 |
12 |
25 |
0 |
3 |
31 |
31 |
|||
200 |
(58) |
5 (2.5) |
0 (0) |
23 (11.5) |
47 (23.5) |
0 (0) |
6 (3.0) |
57 (28.5) |
57 (28.5) |
|||
65 |
100 |
36 |
3 |
- |
0 |
6 |
11 |
0 |
0 |
13 |
13 |
+ |
100 |
35 |
5 |
0 |
3 |
16 |
0 |
2 |
18 |
18 |
|||
200 |
(36) |
8 (4.0) |
0 (0) |
9 (4.5) |
27 (13.5) |
0 (0) |
2 (1.0) |
31 (15.5) |
31 (15.5) |
|||
Positive control (Mitomycin C, 0.05 µg/mL) |
100 |
98 |
0 |
- |
0 |
4 |
13 |
0 |
0 |
16 |
16 |
+ |
100 |
98 |
0 |
2 |
5 |
8 |
0 |
4 |
15 |
16 |
|||
200 |
(98) |
0 |
2 (1.0) |
9 (4.5) |
21 (10.5) |
0 (0) |
4 (2.0) |
31 (15.5) |
32 (16.0) |
|||
Numerical values inside parenthesis shows a mean value.
g: Chromatic gap or chromosome gap
Ctd: chromatid break
Cte: Chromatid exchange
Csb: Chromosome break
Cse: Chromosome exchange
TA: Total aberrant cells excluding gaps
TAG: Total aberrant cells including gaps
Results of the Chromosome Aberration Test With S9-Mix.
Concentration (µg/mL) |
Cells scored |
Cell growth rate (%) |
Number of aberrations (%) |
Number and percentage (%) of cells carrying structural aberrations |
Judgement |
|||||||
No. of polyploids |
Judgement |
g |
ctb |
cte |
csb |
cse |
TA |
TAG |
||||
Untreated control |
100 |
- |
0 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
200 |
- |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|||
Solvent control (physiological saline) |
100 |
100 |
0 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
200 |
(100) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|||
25 |
100 |
94 |
0 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
93 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
200 |
(94) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|||
35 |
100 |
85 |
0 |
- |
0 |
1 |
2 |
0 |
0 |
3 |
3 |
- |
100 |
88 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
|||
200 |
(87) |
0 (0) |
0 (0) |
2 (1.0) |
2 (1.0) |
0 (0) |
0 (0) |
4 (2.0) |
4 (2.0) |
|||
45 |
100 |
67 |
0 |
- |
0 |
4 |
10 |
0 |
5 |
12 |
12 |
+ |
100 |
66 |
0 |
0 |
3 |
9 |
0 |
1 |
10 |
10 |
|||
200 |
(67) |
0 (0) |
0 (0) |
7 (3.5) |
19 (9.5) |
0 (0) |
6 (3.0) |
22 (11.0) |
22 (11.0) |
|||
55 |
100 |
47 |
3 |
- |
1 |
5 |
10 |
0 |
4 |
12 |
12 |
+ |
100 |
42 |
2 |
0 |
1 |
12 |
0 |
2 |
13 |
13 |
|||
200 |
(45) |
5 (2.5) |
1 (0.5) |
6 (3.0) |
22 (11.0) |
0 (0) |
6 (3.0) |
25 (12.5) |
25 (12.5) |
|||
Positive control (N-Nitrosodimethylamine, 0.4 mg/mL) |
100 |
86 |
0 |
- |
1 |
13 |
31 |
0 |
3 |
40 |
40 |
+ |
100 |
91 |
0 |
0 |
8 |
35 |
0 |
2 |
40 |
40 |
|||
200 |
(89) |
0 (0) |
1 (0.5) |
21 (10.5) |
66 (33.0) |
0 (0) |
5 (2.5) |
80 (40) |
80 (40) |
|||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Under the conditions of this study, the results illustrate that the test material has no potency of eliciting micronuclei in vivo when intraperitoneally administrated to male ICR mice at 7.8125, 15.625, 31.25 and 62.5 mg/kg twice at 24-hour interval.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 25 December 2003 to 29 March 2004
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- To investigate the potency of eliciting micronuclei in vivo of the test material, a micronucleus study was conducted using male ICR mice. Mice were intraperitoneally administered with the test material twice at 24-hour interval and the specimens were prepared at 24 hours after the last administration.
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- ICR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 8 weeks old
- Weight at study initiation: Body weight ranges at start of administration were 31.7 - 36.8 g, 32.8 - 37.7 g and 31.6 - 37.7 g in the preliminary, additional test for preliminary and main tests respectively.
- Housing: Six mice were housed in a cage (5 - 6 mice during the quarantine and acclimation period)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 12 days. Additional animals for the preliminary test were quarantined and acclimated for 1 day. General signs were observed every day and the animals were weighed on the next day of receipt and the end of the period.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26 °C (actual value 22 - 23 °C)
- Humidity (%): 40 - 70 % (actual value 57 - 66 %)
- Air changes (per hr): 10 - 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours (7:00 - 19:00) - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: Olive oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Adequate amount of the test material was weighed, suspended with agate mortar gradually adding olive oil to prepare 0.625 w/v% suspension, and then other suspensions (0.3125, 0.15625 and 0.078125 w/v%) were prepared by a serial dilution method. Each formulation was used for administering to the 62.5, 31.25, 15.625 and 7.8125 mg/kg group.
In the preliminary test, adequate amount of the test material was weighed, suspended with agate mortar gradually adding olive oil to prepare 20, 10 and 2.5 w/v% suspensions. Each formulation was used for administering to the 2 000, 1 000, 500 and 350 mg/kg group.
In the additional preliminary test, adequate amounts of the test material was weighed, suspended in agate mortar gradually adding olive oil to prepare 1.25 w/v% suspension and then other suspensions (0.625 and 0.3125 w/v%) were prepared by serial dilution method. Each formulation was used for administering to the 125, 62.5 and 31.25 mg/kg group.
In the main test, adequte amounts of the test material were weighed, suspended in agate mortar gradually adding olive oil to prepare 0.625 w/v% suspension and then other suspensions (0.3125, 0.15625 and 0.078125) were prepared by serial dilution. Each formulation was used for administering to the 62.5, 31.25, 15.625 and 7.8125 mg/kg group.
DOSING METHOD AND ROUTE
Intraperitoneal injection, which is widely used in the micronucleus test, was chosen as the route of administration. The number of doses was set at 24-h intervals. Disposable syringes and needles were used for administration. Dosing volume was calculated individually based on the determined body weight on the day of administration at a rate of 10 mL/kg. - Frequency of treatment:
- The test material was administered twice at 24 h intervals.
- Dose / conc.:
- 62.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 31.25 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 15.625 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 7.812 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 6 animals per dose.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Positive control: Mitomycin C
- Route of administration: Intraperitoneally administered once
- Concentration/ Dose: 2 mg/kg
- Justification for choice of positive control: Widely used in the micronucleus assay.
- Preparation: In both the preliminary and main test, one vial of mitomycin C (2 mg/vial) was dissolved in 5 mL water for injection on the day of use and then diluted 2-fold with physiological saline to prepare formulation for the positive control group (0.2 mg/mL). The formulation of the positive control was kept in well closed umber glass bottles and stored at room temperature until use. - Tissues and cell types examined:
- The potency of eliciting micronuclei and the frequency of total polychromatic erythrocytes of each article were examined.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: In the preliminary test, according to information from the sponsor that the LD50 is > 2 000 mg/kg vial the oral route in rats, 2 000 mg/kg was selected as the highest dose. Other dose levels were set at 1 000, 500 and 350 mg/kg with the common ratio of 2.
Since all animals treated with the test material died by the next day of the first administration of the preliminary test an additional preliminary test was performed.
In the additional preliminary test, the highest dose of the test material was set at 125 mg/kg. Other dose levels were set at 62.5 and 31.25, with the common ratio of 2. All animals of the 125 mg/kg group died by the next day of the first administration and one animal of the 62.5 mg/kg group died after the second administration. In animals that survived, there were bradypnea, decrease in locomotor activity, abnormal fur, writhing etc. Bradypnea and writhing were also noted at the 31.25 mg/kg group. Tendency to suppress body weight changes were noted in the 31.25 mg/kg group.
In observation of the specimens, there was no tendency to depend on dose, nor time-point to prepare the specimens in the frequency of MNPCE and a total of PCE/RBC in the test material groups.
Based on the above results, the time to prepare the specimens was set at 24 h after the last administration the highest dose was set at 62.5 mg/kg and other dose levels were set at 31.25, 15.625 and 7.8125 mg/kg with a common ratio of 2. In addition, two groups with the negative control (olive oil) and the positive control group (MMC, 2 mg/kg) were set.
TREATMENT AND SAMPLING TIMES: Mice survived until the time to prepare the specimens were euthanized by cervical vertebra dislocation method.
DETAILS OF SLIDE PREPARATION: To collect bone marrow cells, right femur was washed with a little volume of foetal calf serum (about 1.0 mL/femur). The bone marrow cell suspension was centrifuged at 1 000 rpm for 5 minutes and the supernatant was discarded. The bone marrow cells in the tube were well stirred with a Pasteur pipette to get homogeneous cell suspension. A drop of the suspension was dripped on a slide glass and the smear was prepared using a cover glass. The specimens were prepared in duplicate. The specimens were dried at room temperature, fixed with methanol for 5 minutes and stained with 3 % Giemsa solution in Sorensen-phosphate buffer (1/15 M, pH 6.8) for 30 minutes. The stained specimens were washed with Sorensen-phosphate buffer, dipped in 0.004 % citric acid solution for several second, washed with distilled water, and then dried at room temperature.
METHOD OF ANALYSIS: One of the specimens prepared 2 slides were observed by blind test method (the specimens were identified by quarantine No.) with a microscope equipped ocular lenses (x 10) and an oil immersion objective lens (x 100). To evaluate the depression of proliferation of erythroblast of the test article, a ratio of total polychromatic erythrocytes count to total erythrocytes (PCE / RBC) was calculated by counting 1000 erythrocytes in each animal. A ratio of micronucleated polychromatic erythrocytes (MNPCE) was calculated by counting 2 000 total polychromatic erythrocytes (total PCE) in each animal. - Evaluation criteria:
- Acceptance criteria were as follows:
1) The frequency of MNPCE in the negative control group in the main test was lower than the upper limit of frequency in the background data collected from the negative control groups in the facility (0.11 + 0.09 %, mean + 3 x standard deviation).
2) The frequency of NPCE in the positive control group was higher than the upper limit of frequncy in the background data. - Statistics:
- Numbers of MNPCE were statistically analysed by binominal test (one-sided 5 %) according to a table of Kastenbaum and Bowman (Mutation Res., 9: 527-549, I 970) using SAS Release 6.1 2 (SAS Institute Inc.). To evaluate depression of proliferation of the bone marrow cells, the ratio of total polychromatic erythrocytes to total erythrocytes were analyzed by Bartlett's test for homogeneity of variances (significant level, one-sided: 5 and 1 %). Because the variances were homogeneous, Dunnett's test was perfomed to compare each of the treated group means with the mean of the negative control group (significant level, two-sided, 5 and 1 %). The Statistical analysis was performed using StatLight® (Yukms Corp.).
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Death occurred in 1 animal (1/6) of the 62.5 mg/kg group on the next day of the first administration. Bradypnea (6/6), decrease in locomotor activity (6/6), writhing (2/6), abnormal fur (5/6) and loose stool (2/6) were noted in the same group. No abnormalities were noted in other groups. Depression of body weight change was noted in 62.5 mg/kg. No marked changes were noted in other groups in body weight changes.
In the positive control group, which received MMC (2 mg/kg) intraperitoneally once, the frequency of micronucleated polychromatic erythrocytes (MNPCE) at 24 hours after administration was 4.41 ± 1.06 %, which was much higher than that of the negative control group (0.13 ± 0.04 %). Number of MNPCE (529) was judged as positive by binominal test according to the table of Kastenbaum and Bowman. The frequency of MNPCE of the group was higher than the upper limit of the frequency in the background data of the negative control. On the other hand, the frequency of MNPCE in the negative control group was lower than the upper limit of the frequency in the background data. Since the results met the acceptance criteria, the study was considered to be carried out properly.
The frequency of MNPCE was evaluated 24 hours after the last administration of the test material of each dosage (administration was perfomed twice at 24-hour interval). The frequency of 62.5, 31.25, 15.625 and 7.8125 mg/kg groups were 0.20 ± 0.06, 0.13 ± 0.07, 0.13 ± 0.06 and 0.12 ± 0.05 %, respectively and they were lower than the upper limit of the frequency in the background data. These results were judged as negative by binominal test according to the table of Kastenbaum and Bowman.
There was slight depression of proliferation in the ratio of total polychromatic erythrocytes to total erythrocytes in the 62.5 mg/kg group by comparing to the negative control group. - Conclusions:
- Interpretation of results: Negative
Under the conditions of this study, the results illustrate that the test material has no potency of eliciting micronuclei in vivo when intraperitoneally administrated to male ICR mice at 7.8125, 15.625, 31.25 and 62.5 mg/kg twice at 24-hour interval. - Executive summary:
A micronucleus study was conducted using male ICR mice to investigate the potency of eliciting micronuclei in vivo by the test material.
Mice were intraperitoneally administered with 62.5, 31.25, 15.625, 7.8125 or 0 mg/kg (negative control) of the test material twice at 24 hour intervals and the specimens were prepared at 24 h after the last administration. The animals in the positive control group were intraperitoneally administered with 2 mg/kg Mitomycin C once and the specimens were prepared at 24 h after administration. The potency of eliciting micronuclei and the frequency of total polychromatic erythrocytes of each article were examined.
In the 62.5 mg/kg group death occurred in 1 animal (1/6). In the surviving animals, decreased body weights were noted. Bradypnea, decrease in locomotor activity and abnormal fur were also noted in general signs. The frequency of micronucleated polychromatic erythrocytes were lower than upper limit of the frequency in the background data collected from the negative control group and was judged negative in statistical analysis. In the positive control group the frequency was higher than the upper limit of the frequency in the background data and was judged as positive in statistical analysis.
In the 62.5 mg/kg group there were statistically significant differences in the ratio of total polychromatic erythrocytes to total erythrocytes from the negative control group and slight depression of proliferation of erythrocytes were recognised. However, this change was considered to be associated with the toxic changes attributed to administration of massive doses since occasion of death and decrease of bodyweight and other toxic changes in the surviving animals was noted in the same group.
The results suggest that the test material has no potency of eliciting micronuclei in vivo when administered intraperitoneally to male ICR mice under the conditions of this study.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In Vitro Ames: Kato (2003)
The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted under GLP conditions. In this study, the test material was assessed for its potential to induce gene mutations according to the pre-incubation test (both experiments) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in duplicate. The test item was tested at the following concentrations: experiment I (preliminary test): 5, 20, 78, 313, 1250, 5000 µg/plate and experiment II: 313, 625, 1 250, 2 500, 5 000 µg/plate.
Neither toxic effect of the test material nor two-fold or more increase in the number of the revertant colonies with dose-relativity compared with the negative control was observed in any bacterial strains at any dose level regardless of the presence or the absence of metabolic activation. Moreover, precipitation of the test material was not detected in any dose levels.
Under the conditions of this study the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, it is concluded from the foregoing results that the mutagenic activity of the test substance is considered negative in both the presence and absence of metabolic activation.
In Vitro Chromosome Aberration: Yokota (2003)
The genotoxic potential of the test material was investigated in a study which was conducted under GLP conditions and following a method which was similar to that which is outlined in the standardised guideline OECD 473.
Thisin vitrochromosome aberration test in cultured mammalian cells (CHL/IU cells) was performed to assess the potential of the test material to induce chromosome aberrations.
The maximum concentration used in cell growth inhibition test was 3.580 mg/mL. Physiological saline was chosen as solvent for this study.
The precipitation of the test substance in culture medium was observed at more than 1.790 mg/mL in the cell growth inhibition test with and without a metabolic activation system (hereinafter referred to as “with S9 mix” and “without S9 mix”).
In the results of the cell growth inhibition test, the concentrations of 50 % cell growth inhibition in short-term exposure experiments without S9 mix and with S9 mix were 0.052 mg/mL and 0.045 mg/mL. Therefore, the maximum concentration of test substance without S9 mix was 65 μg/mL, and 4 applied concentrations which were prepared with dilution by a common difference of 10 μg/mL were set up and examined. In with S9 mix, the maximum concentration of test substance was 55 μg/mL, and 4 applied concentrations, prepared with dilution by a common difference of 10 μg/mL, were set up and examined.
In the results of the chromosome aberration test both with and without S9 mix, the frequencies of cells carrying polyploidy were less than 5 % at any concentration. However, the frequencies of cells carrying structural chromosome aberrations without S9 mix increased to 28.5 and 15.5 % at the dose levels of 55 and 65 μg/mL, respectively. The results of that with S9 mix increased to 11.0 and 12.5 % at the dose levels of 45 and 55 μg/mL, respectively. Therefore, the results were judged positive.
In the positive control group in each experiment, the frequencies of cells carrying structural chromosome aberrations were suitable values showing that the experiments were performed appropriately. As the results of the short-term exposure experiments were judged positive, the continuous exposure experiments were not performed.
Based on the above results, it was concluded in the study report that the test material induces chromosome aberrations in the cell line of Chinese hamster fibroblast in lung (CHL/IU cells). However, genotoxicity observed with zinc is secondary to effects on other cellular components, and not due to direct interaction of zinc with DNA, or oxidative stress. Excess zinc can lead to dysregulation of cell function because zinc is an important intracellular messenger molecule leading to cytotoxicity and potentially exacerbating secondary effects of zinc on the genome. The effects seen in the Yokota (2004) are therefore likely to be due to concentrations of zinc in the cultures with which the cells are unable to maintain intracellular zinc homeostasis. This gives rise to the distinct threshold above which cytotoxicity and the chromosome aberrations appeared in Yokota. The study by Hargitai with the sodium salt of the test material showed the result seen was not likely to be attributable to the organic part of the molecule, which also lacks any structural alerts for genotoxicity
The test material should not therefore be considered a direct non-threshold genotoxin, and any apparent genotoxic effects should be attributed as secondary effects of zinc excess on the cells in question.
In Vitro Chromosome Aberration: Hargitai (2013) Read-Across
The clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with the standardised guideline OECD 473, EU Method B.10 and EPA OPPTS 870.5375.
Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. Both experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period, or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.
Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication of polyploidy or endoreplication.
The test material is therefore considered not to be clastogenic in this test system.
In Vivo Micronucleus Assay: Aoyama (2004)
A micronucleus study was conducted using male ICR mice to investigate the potency of eliciting micronuclei in vivo by the test material.
Mice were intraperitoneally administered with 62.5, 31.25, 15.625, 7.8125 or 0 mg/kg (negative control) of the test material twice at 24 hour intervals and the specimens were prepared at 24 h after the last administration. The animals in the positive control group were intraperitoneally administered with 2 mg/kg Mitomycin C once and the specimens were prepared at 24 h after administration. The potency of eliciting micronuclei and the frequency of total polychromatic erythrocytes of each article were examined.
In the 62.5 mg/kg group death occurred in 1 animal (1/6). In the surviving animals, decreased body weights were noted. Bradypnea, decrease in locomotor activity and abnormal fur were also noted in general signs. The frequency of micronucleated polychromatic erythrocytes were lower than upper limit of the frequency in the background data collected from the negative control group and was judged negative in statistical analysis. In the positive control group the frequency was higher than the upper limit of the frequency in the background data and was judged as positive in statistical analysis.
In the 62.5 mg/kg group there were statistically significant differences in the ratio of total polychromatic erythrocytes to total erythrocytes from the negative control group and slight depression of proliferation of erythrocytes were recognised. However, this change was considered to be associated with the toxic changes attributed to administration of massive doses since occasion of death and decrease of bodyweight and other toxic changes in the surviving animals was noted in the same group.
The results suggest that the test material has no potency of eliciting micronuclei in vivo when administered intraperitoneally to male ICR mice under the conditions of this study.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to mutagenicity.
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