Registration Dossier
Registration Dossier
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EC number: 207-668-4 | CAS number: 488-10-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Negative results in Salmonella typhimurium
TA 98, TA 100, TA 102, TA 1535 and TA 1537, with and without metabolic
activation (OECD TG 471, Bacterial Reverse Mutation Assay, GLP).
Negative results in human lymphocytes, with and without metabolic
activation (OECD TG 487, In vitro Mammalian Cell Micronucleus Test, GLP)
Negative results in an in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of the test substance to cause gene mutation (OECD TG 476, GLP)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- January 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study done to guideline, in compliance with GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information):
negative
During the described mutagenicity test and under the experimental conditions reported, Jasmone Cis did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. - Executive summary:
During the described mutagenicity test and under the experimental conditions reported, Jasmone Cis did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 April 2014 - 27 January 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- Protocol deviation : In the cytogenetic assays 1A, 1B, 1C and 1D the positive control cultures were exposed to only one concentration of Cyclophosphamide (15 μg/ml).
Evaluation: Since a clear positive result was obtained at a dose level of 15 μg/ml, adding an
additional higher dose of Cyclophosphamide would have given no additional information.
The study integrity was not adversely affected by the deviation.
Standard operating procedures deviation : There were no deviations from standard operating procedures that affected the integrity of the study. - Deviations:
- yes
- Remarks:
- There were no deviations from standard operating procedures that affected the integrity of the study.
- Qualifier:
- according to guideline
- Guideline:
- other: (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.49 “InVitro Mammalian Cell Micronucleus Test". OJ of the EU No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.
- Version / remarks:
- Protocol deviation : In the cytogenetic assays 1A, 1B, 1C and 1D the positive control cultures were exposed to only one concentration of Cyclophosphamide (15 μg/ml).
Evaluation: Since a clear positive result was obtained at a dose level of 15 μg/ml, adding an
additional higher dose of Cyclophosphamide would have given no additional information.
The study integrity was not adversely affected by the deviation.
Standard operating procedures deviation : There were no deviations from standard operating procedures that affected the integrity of the study. - Deviations:
- yes
- Remarks:
- There were no deviations from standard operating procedures that affected the integrity of the study.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Blood was collected from healthy adult, non-smoking, male volunteers (aged < 35 years)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Dose range finding test: 17, 52, 164, 512 and 1642 μg test substance/ml culture medium
First cytogenetic assay: With S9-mix: 100, 400, 450, 465, 480, 495, 510, 525, 540, 555, 570, 585 and 600 μg/mL culture medium
scoring of micronuclei: With S9-mix: 100, 450 and 495 μg/mL culture medium
Second cytogenetic assay: Without S9-mix : 10, 50, 70, 90, 110, 130 and 150 μg test item/mL culture medium
scoring of micronuclei: Without S9-mix : 10, 50 and 70 μg test item/mL culture medium - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Remarks:
- vehicle
- Negative solvent / vehicle controls:
- yes
- Remarks:
- for positive controls: HBSS without calcium and magnesium.
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Colchicine (CAS no. 64-86-8), Without metabolic activation
- Details on test system and experimental conditions:
- Test system: Cultured peripheral human lymphocytes
DURATION
- Culture period: 46 ± 2 hours
- Exposure duration: 24h (without metabolic activation), 3h with metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 27 hours - Evaluation criteria:
- A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision. - Statistics:
- Chi-square Test
- Key result
- Species / strain:
- lymphocytes: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The number of mono- and binucleated cells with micronuclei found in the solvent control was within the historical control data range and was less than 10 per 2000 mono- or binucleated cells. The test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
See table 1A, 1B and 2.
Dose range finding results:
see attachement CBPI Pages from MU_FINAL Report_In vitro Micronucleus_PHL_REACH - Conclusions:
- Finally, it is concluded that this test is valid and that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 September 2017 to 23 October 2017
- 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)
- Version / remarks:
- OECD Guidelines for Testing of Chemicals No. 476, "In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes" (adopted 29 July 2016)
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Commission Regulation (EC) No 440/2008 of 30 May 2008, B.17. "In vitro Mammalian Cell Gene Mutation Test”, (Official Journal L 142, 31/05/2008)
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: mammalian cell gene mutation
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (hprt) enzyme locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO K1: Sub-line (K1) of Chinese hamster ovary cell line CHO
ATCC No.: CCL-61
Lot No.: 58244452
Date of Arrival: 10 August 2010
Date of Reconstitution: 03 January 2011
Supplier: American Type Culture Collection (Manassas, Virginia, United States)
The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased from American Type Culture Collection (ATCC). Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 22 April 2016. Cells were stored as frozen stocks in a liquid nitrogen tank.
Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium and incubated at 37 °C (± 0.5 C) in a humidified atmosphere (5± 0.3% CO2 in air).
When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1
5-hour treatment in the presence of S9-mix: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL
5-hour treatment in the absence of S9-mix: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL
Assay 2
5-hour treatment in the presence of S9-mix: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL
24-hour treatment in the absence of S9-mix: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL
Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. For the treatments in the preliminary toxicity test, a formulation of 200 mg/mL was prepared using the selected vehicle (solvent) and lower test concentrations were prepared by serial dilutions. The highest test concentration in the preliminary test was 2000 μg/mL (the maximal recommended concentration). - Vehicle / solvent:
- DMSO was used as the vehicle (solvent) of the test item in this study.
Data of the chemical used for vehicle (solvent) control of the study are shown below:
Name: Dimethyl sulfoxide
Abbreviation: DMSO
Supplier: Sigma-Aldrich Co./ VWR
Lot No.: SZBG1310V / STBG8411/ 16F304002
Expiry date: 25 April 2019/ 29 February 2020/ 31 May 2021
Storage conditions: Room temperature, under N2 - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- TEST PROCEDURE
Principles of dose selection (Preliminary Toxicity Test)
Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. For the treatments in the preliminary toxicity test, a formulation of 200 mg/mL was prepared using the selected vehicle (solvent) and lower test concentrations were prepared by serial dilutions. The highest test concentration in the preliminary test was 2000 μg/mL (the maximal recommended concentration*).
*Note: For a relatively non-cytotoxic compound the maximum concentration is 2 μL/mL, 2 mg/mL or 10 mM, whichever is the lowest. When a test item is not of defined composition (e.g. substance of unknown or variable composition, complex reaction products or biological materials), the top concentration may need to be higher (e.g. 5 mg/mL) in the absence of sufficient toxicity, to increase the concentration of each of the components. For relatively insoluble substances, the highest dose is a concentration above the limit of solubility in the final culture medium at the end of the treatment.
Where cytotoxicity occurs, the highest concentration selected is expected to result in approximately 10-20% relative survival (%RS). The lower test concentrations are normally separated by factors of two. The higher test concentrations may be spaced more closely in order to increase the chance of producing the required level of toxicity.
In the preliminary experiment, a 5-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments.
Treatment of cell cultures was performed as described below for the main mutation assays. However, single cultures were tested and positive controls were not included.
Following treatments (as cytotoxicity was observed on Day 1), cell number in the samples was adjusted to 2x105 cells/mL after counting and cells (10 mL cell suspension) were transferred to dishes for growth some additional days. After the incubation period, cell concentrations were determined using a haemocytometer on Day 3, 6 and 8.
Precipitation of the test item in the final culture medium was visually examined at the beginning and end of the treatments. The pH and osmolality of the treatment medium at the end of the treatment was also determined.
Mutation Assays
In Assay 1, 5-hour treatment was performed with and without metabolic activation (in the presence and absence of S9-mix). In Assay 2, 5-hour treatment was performed with metabolic activation (in the presence of S9-mix) and 24-hour treatment without metabolic activation (in the absence of S9-mix).
Treatment of the cells
For the 5-hour treatments, at least 2x10^6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation). A suitable volume (100 μL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures. After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium (10-60 mL) and incubated for 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10^5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
For the 24-hour treatment, at least 2x10^6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and incubated for approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5% serum (F12-5). A suitable volume (100 μL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 24-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10^5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
Duplicate cultures were used for each treatment except of 24-hour treatment in case of Assay 2 where due to the observed excessive cytotoxicity there were not duplicate in case of the 450 μg/mL. Solubility of the test item in the cultures was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment.
Plating for survival
Following adjustment of the cultures to 2x10^5 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium.
Five mL suspension (200 cells/dish) per each culture were plated into 3 parallel dishes (diameter was approx. 60 mm). The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.
Expression period
Cultures were maintained in dishes for 7 days, during which time the HPRT-mutation was expressed. During this expression period, the cultures were sub-cultured and maintained at 2x10^5 cells/dish twice (on Days 3, 6 and 8, or on Days 4, 6 and 8), to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell density was determined by haemocytometer and cells were plated for viability and for selection of the mutant phenotype.
Plating for viability
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x105 cells/mL, then further diluted to 40 cells/mL using F12-10 medium.
Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.
Plating for selection of the mutant phenotype (6-TG resistance)
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10^5 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approx. 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 μg/mL 6-thioguanine (abbreviation: 6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 μg/mL) to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.
Fixation and staining of colonies
After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted. - Rationale for test conditions:
- In accordance with the test guidelines.
- Evaluation criteria:
- Acceptance criteria
The assay was considered valid if all the following criteria are met:
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test item concentrations in duplicate cultures were presented.
Evaluation criteria
The test item was considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary). Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. In cases with survival lower than 10%, extreme caution is taken in the interpretation.
According to the relevant OECD guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response. - Statistics:
- The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If the obtained result were significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.
The mutation frequencies were statistically analyzed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency). - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY EXPERIMENT
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 μg/mL (the recommended maximum concentration).
Insolubility and cytotoxicity were detected in the preliminary experiment. The concentrations selected for the main experiments were based on the cytotoxicity data to cover the range from cytotoxicity to no or little cytotoxicity according to the relevant OECD guideline. Lower test concentrations were separated by factor of two, but more closely spaced concentration were selected in the expected cytotoxic concentration range. Seven concentrations were selected for the main experiments.
MUTATION ASSAYS
In the mutation assays, cells were exposed to the test item for 5 hours with and without metabolic activation system (±S9-mix) or for 24 hours without metabolic activation system (-S9-mix) then the cells were plated for determination of survival and in parallel sub-cultured without test item for 7 days to allow the expression of the genetic changes (if any occurred). At the end of the expression period, cells were allowed to grow and form colonies in culture dishes with and without selective agent (6-TG) for determination of mutations and viability.
Assay 1
In Assay 1, a 5-hour treatment with metabolic activation (in the presence of S9-mix) and a 5-hour treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-hour treatment in the presence of S9-mix, the following concentrations were examined: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL.
For the 5-hour treatment in the absence of S9-mix, the following concentrations were examined: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL.
In Assay 1, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 13.6% and 10% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
In the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 5.8% and 31% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. Statistically significant increase (at p<0.01 level) was observed in this experiment at 350 μg/mL concentration, although the observed value was within the general historical control range.
Furthermore, the observed mutant frequencies (8.5 x 10^-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment).
Assay 2
In Assay 2, 5-hour treatment with metabolic activation (in the presence of S9-mix) and 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-hour treatment in the presence of S9-mix, the following concentrations were examined: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL.
For 24-hour treatment in the absence of S9-mix, the following concentrations were examined: 450, 400, 350, 300, 150, 75 and 37.5 μg/mL.
In Assay 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 23.2% and 15% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results seen in Assay 1 with metabolic activation.
In the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL and the following concentration 400 μg/mL showed a relative survival of 0.1%, 2% and 7.2%, 23% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results without metabolic activation.
The other sporadic, statistically non-significant differences were examined for consistency; none of them were repeatable when comparing Assay 1 and Assay 2.
Furthermore, all the observed mutation frequency values were within the general historical control range. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between treated samples and negative (vehicle) controls.
VALIDITY OF THE MUTATION ASSAYS
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays, and the observed values were in the expected range (5-20 x 10^-6) as shown in the OECD No. 476 guideline.
The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays.
The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays.
The tested concentration range in the study was considered to be adequate as the defined acceptance criteria of the relevant OECD regarding cytotoxicity produced by the highest evaluated concentrations (approximately 80-90 % toxicity, i.e. approximately 10-20 % relative survival*) were considered to be fulfilled.
Seven test item concentrations were evaluated in duplicate in each experiment.
The overall study was considered valid. - Conclusions:
- The HPRT Assay with the test item performed on CHO K1 Chinese hamster ovarian cells was considered to be valid and reflect the real potential of the test item to cause mutations in the cultured mammalian cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies in the presence and did not result in a biologically significant dose-dependent increase in mutation frequencies in the absence of a rat metabolic activation system (S9) in this study.
In conclusion, no mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay. - Executive summary:
An in vitro mammalian cell assay [1-2] was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of the test substance to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix).
DMSO was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1
5-hour treatment in the presence of S9-mix:
450, 400, 350, 300, 150, 75 and 37.5 μg/mL
5-hour treatment in the absence of S9-mix:
450, 400, 350, 300, 150, 75 and 37.5 μg/mL
Assay 2
5-hour treatment in the presence of S9-mix:
450, 400, 350, 300, 150, 75 and 37.5 μg/mL
24-hour treatment in the absence of S9-mix:
450, 400, 350, 300, 150, 75 and 37.5 μg/mL
In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.
In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 13.6% and 10% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 5.8% and 31% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. Statistically significant increase (at p<0.01 level) was observed in this experiment at 350 μg/mL concentration, although the observed value was within the general historical control range.
Furthermore, the observed mutant frequencies (8.5 x 10^-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10 ^-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment).
In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 23.2% and 15% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results seen in Assay 1 with metabolic activation.
In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL and the following concentration 400 μg/mL showed a relative survival of 0.1%, 2% and 7.2%, 23% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results without metabolic activation.
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. Seven evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the cytotoxic range in each test). The overall study was considered to be valid.
In conclusion, no mutagenic effect of the test substance was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.
Referenceopen allclose all
During the described mutagenicity test and under the experimental conditions reported, Jasmone Cis did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Table 1 A and B: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with the test substance in the first cytogenetic assay:
Table 1A: Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (μg/ml) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei 1) |
Number of binucleated cells with micronuclei 1) |
|||||
|
|
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
|
A |
B |
A+B |
A |
B |
A+B |
|||
0 |
0 |
2 |
2 |
4 |
2 |
5 |
7 |
|
100 |
0 |
2 |
0 |
2 |
2 |
6 |
8 |
|
400 |
34 |
1 |
0 |
1 |
1 |
0 |
1 |
|
440 |
57 |
1 |
2 |
3 |
1 |
1 |
2 |
|
0.25 MMC-C |
30 |
0 |
2 |
2 |
32 |
47 |
79*** |
|
0.1 Colch |
72 |
30 |
50 |
80*** |
8 |
13 |
21** |
|
Table 1B: With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (μg/ml) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei 1) |
Number of binucleated cells with micronuclei 1) |
|||||
|
|
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
|
A |
B |
A+B |
A |
B |
A+B |
|||
0 |
0 |
0 |
0 |
0 |
8 |
3 |
11 |
|
100 |
-2 |
3 |
3 |
6 |
2 |
2 |
4 |
|
450 |
28 |
1 |
3 |
4 |
6 |
1 |
7 |
|
495 |
58 |
1 |
0 |
1 |
5 |
11 |
16 |
|
15 CP |
39 |
2 |
1 |
3 |
21 |
14 |
35*** |
|
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000-1000 bi- and mononucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B.
Table 2: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with the test item in the second cytogenetic assay:
Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (μg/ml) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei 1) |
Number of binucleated cells with micronuclei 1) |
||||
|
|
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
A |
B |
A+B |
A |
B |
A+B |
||
0 |
0 |
1 |
0 |
1 |
3 |
2 |
5 |
10 |
3 |
0 |
0 |
0 |
2 |
3 |
5 |
50 |
28 |
2 |
1 |
3 |
5 |
5 |
10 |
70 |
57 |
1 |
1 |
2 |
3 |
4 |
7 |
0.15 MMC-C |
47 |
2 |
0 |
2 |
24 |
24 |
48*** |
0.05 Colch |
96 |
40 |
55 |
95*** |
12) |
02) |
1 |
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B.
2) 98 and 85 binucleated cells were evaluated for the presence of micronuclei, respectively.
MMC-C: Mitomycin C
Colch: Colchicine
CP: Cyclophosphamide
Preliminary Toxicity Test Results
5-hour treatment in the presence of metabolic activation
Test item concentration |
Cell number, cells/mL (Relative Survival*, %) |
Observations At the beginning / after treatment |
|||
After treatment (Day 1) |
Day 3 |
Day 6 |
Day 8 |
||
Untreated control |
7.43E+06 (132) |
8.00E+06 (100) |
5.90E+06 (83) |
6.20E+06 (84) |
Normal / normal (pH: 7.0; osm: 289 mmol/kg) |
Negative (vehicle) control |
5.63E+06 (100) |
8.00E+06 (100) |
7.10E+06 (100) |
7.40E+06 (100) |
Normal / normal (pH: 7.0; osm: 437 mmol/kg) |
2000 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#, oily drops, oily film / discoloured medium#, oily drops, oily medium (pH: 7.0, osm: 395 mmol/kg) |
1000 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#, oily drops, oily film / discoloured medium#(pH: 7.0, osm: 412 mmol/kg) |
500 μg/mL |
1.50E+05 (3) |
0.00E+00 (0) |
-- |
-- |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 418 mmol/kg) |
250 μg/mL |
3.70E+06 (66) |
7.90E+06 (99) |
6.50E+06 (92) |
5.60E+06 (76) |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 425 mmol/kg) |
125 μg/mL |
4.73E+06 (84) |
9.80E+06 (123) |
6.20E+06 (87) |
4.90E+06 (66) |
Normal / discoloured medium#(pH: 7.0; osm 428 mmol/kg) |
62.5 μg/mL |
5.35E+06 (95) |
8.10E+06 (101) |
5.70E+06 (80) |
5.50E+06 (74) |
Normal / discoloured medium#(pH: 7.0; osm 434 mmol/kg) |
31.25 μg/mL |
6.50E+06 (116) |
9.10E+06 (114) |
6.10E+06 (86) |
5.20E+06 (70) |
Normal / discoloured medium#(pH: 7.0; osm 437 mmol/kg) |
15.625 μg/mL |
5.65E+06 (100) |
8.10E+06 (101) |
7.60E+06 (107) |
5.60E+06 (76) |
Normal / discoloured medium#(pH: 7.0; osm 438 mmol/kg) |
7.813 μg/mL |
6.43E+06 (114) |
8.40E+06 (105) |
6.00E+06 (85) |
5.80E+06 (78) |
Normal / normal (pH: 7,0; osm: 434 mmol/kg) |
3.906 μg/mL |
5.73E+06 (102) |
8.00E+06 (100) |
6.40E+06 (90) |
5.50E+06 (74) |
Normal / normal (pH: 7.0; osm: 436 mmol/kg) |
*: compared to the negative (vehicle) control (Dimethyl sulfoxide) #: minimal amount
osm: osmolality
5-hour treatment in the absence of metabolic activation
Test item concentration |
Cell number, cells/mL (Relative Survival*, %) |
Observations At the beginning / after treatment |
|||
After treatment (Day 1) |
Day 3 |
Day 6 |
Day 8 |
||
Untreated control |
8.33E+06 (112) |
8.20E+06 (96) |
6.90E+06 (111) |
6.10E+06 (105) |
Normal / normal (pH: 7.0; osm: 288 mmol/kg) |
Negative (vehicle) control |
7.43E+06 (100) |
8.50E+06 (100) |
6.20E+06 (100) |
5.80E+06 (100) |
Normal / normal (pH: 7.0; osm: 429 mmol/kg) |
2000 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#, oily drops, oily film / discoloured medium#, oily drops, precipitate (pH: 7.0, osm: 369 mmol/kg) |
1000 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#, oily drops, oily film#/ discoloured medium#, precipitate (pH: 7.0, osm: 408 mmol/kg) |
500 μg/mL |
1.25E+05 (2) |
0.00E+00 (0) |
-- |
-- |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 417 mmol/kg) |
250 μg/mL |
5.35E+06 (72) |
8.60E+06 (101) |
5.80E+06 (94) |
6.70E+06 (116) |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 431 mmol/kg) |
125 μg/mL |
7.13E+06 (96) |
7.60E+06 (89) |
7.00E+06 (113) |
5.80E+06 (100) |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 427 mmol/kg) |
62.5 μg/mL |
8.43E+06 (113) |
7.40E+06 (87) |
6.10E+06 (98) |
5.60E+06 (97) |
Normal / discoloured medium#(pH: 7.0; osm 432 mmol/kg) |
31.25 μg/mL |
7.45E+06 (100) |
8.40E+06 (99) |
6.50E+06 (105) |
5.80E+06 (100) |
Normal / discoloured medium#(pH: 7.0; osm 430 mmol/kg) |
15.625 μg/mL |
8.18E+06 (110) |
8.20E+06 (96) |
6.50E+06 (105) |
5.20E+06 (90) |
Normal / discoloured medium#(pH: 7.0; osm 437 mmol/kg) |
7.813 μg/mL |
7.23E+06 (97) |
8.20E+06 (96) |
6.40E+06 (103) |
4.60E+06 (79) |
Normal / normal (pH: 7,0; osm: 439 mmol/kg) |
3.906 μg/mL |
8.08E+06 (109) |
7.10E+06 (84) |
6.10E+06 (98) |
7.20E+06 (124) |
Normal / normal (pH: 7.0; osm: 438 mmol/kg) |
*: compared to the negative (vehicle) control (Dimethyl sulfoxide) #: minimal amount
osm: osmolaity
24-hour treatment in the absence of metabolic activation
Test item concentration |
Cell number, cells/mL (Relative Survival*, %) |
Observations At the beginning / after treatment |
|||
After treatment (Day 1) |
Day 3 |
Day 6 |
Day 8 |
||
Untreated control |
7.73E+06 (99) |
8.00E+06 (86) |
5.10E+-6 (86) |
6.20E+06 (127) |
Normal / normal (pH: 7.0; osm: 292 mmol/kg) |
Negative (vehicle) control |
7.83E+06 (100) |
9.30E+06 (100) |
5.90E+06 (100) |
4.90E+06 (100) |
Normal / normal (pH: 7.0; osm: 438 mmol/kg) |
2000 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#, oily drops, oily film / discoloured medium, precipitate (pH: 7.0, osm: 408 mmol/kg) |
1000 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#, oily drops, oily film#/ discoloured medium, precipitate#(pH: 7.0, osm: 414 mmol/kg) |
500 μg/mL |
0.00E+00 (0) |
-- |
-- |
-- |
Discoloured medium#/ discoloured medium (pH: 7.0, osm: 427 mmol/kg) |
250 μg/mL |
4.05E+06 (52) |
5.40E+06 (58) |
6.30E+06 (107) |
7.70E+06 (157) |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 434 mmol/kg) |
125 μg/mL |
5.25E+06 (67) |
6.50E+06 (70) |
5.90E+06 (100) |
5.50E+06 (112) |
Discoloured medium#/ discoloured medium#(pH: 7.0, osm: 444 mmol/kg) |
62.5 μg/mL |
7.53E+06 (96) |
6.30E+06 (69) |
6.90E+06 (117) |
6.70E+06 (137) |
Normal / discoloured medium#(pH: 7.0; osm 445 mmol/kg) |
31.25 μg/mL |
7.83E+06 (100) |
7.30E+07 (78) |
6.70E+06 (114) |
5.10E+06 (104) |
Normal / discoloured medium#(pH: 7.0; osm 442 mmol/kg) |
15.625 μg/mL |
7.15E+06 (91) |
8.40E+06 (90) |
5.60E+06 (95) |
6.20E+06 (127) |
Normal / discoloured medium#(pH: 7.0; osm 444 mmol/kg) |
7.813 μg/mL |
7.95E+06 (102) |
8.10E+06 (87) |
6.60E+06 (112) |
5.80E+06 (118) |
Normal / discoloured medium#(pH: 7.0; osm 445 mmol/kg) |
3.906 μg/mL |
8.45E+06 (108) |
8.50E+06 (91) |
5.50E+06 (93) |
5.70E+06 (116) |
Normal / discoloured medium#(pH: 7.0; osm 446 mmol/kg) |
*: compared to the negative (vehicle) control (Dimethyl sulfoxide) #: minimal amount
osm: osmolality
Summarized Survival Results of Assay 1
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Relative Survival (%) |
Total number of colonies |
Cloning Efficiency (CE) |
Relative Survival (%) on plates |
+ |
5 |
A1 |
450 μg/mL |
13.6 |
105 |
0.088 |
10 |
400 μg/mL |
29.8 |
307 |
0.256 |
30 |
|||
350 μg/mL |
29.2 |
348 |
0.290 |
34 |
|||
300 μg/mL |
47.3 |
447 |
0.373 |
43 |
|||
150 μg/mL |
57.9 |
960 |
0.800 |
92 |
|||
75 μg/mL |
83.2 |
1028 |
0.857 |
99 |
|||
37.5 μg/mL |
104.0 |
1031 |
0.859 |
99 |
|||
Negative control |
100.0 |
1038 |
0.865 |
100 |
|||
Untreated control |
128.1 |
985 |
0.821 |
95 |
|||
Positive control (DMBA) |
44.0 |
34 |
0.028 |
3 |
|||
- |
5 |
A1 |
450 μg/mL |
5.8 |
384 |
0.320 |
31 |
400 μg/mL |
25.5 |
593 |
0.494 |
48 |
|||
350 μg/mL |
51.7 |
839 |
0.699 |
68 |
|||
300 μg/mL |
71.0 |
962 |
0.802 |
78 |
|||
150 μg/mL |
75.6 |
1107 |
0.923 |
89 |
|||
75 μg/mL |
91.9 |
1081 |
0.901 |
87 |
|||
37.5 μg/mL |
108.6 |
1090 |
0.908 |
88 |
|||
Negative control |
100.0 |
1239 |
1.033 |
100 |
|||
Untreated control |
115.3 |
1171 |
0.976 |
95 |
|||
Positive control (EMS) |
76.7 |
660 |
0.550 |
53 |
A1 = Assay 1
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 μg/mL
- = in the absence of S9-mix EMS = Ethyl methanesulfonate, 0.4 μL/mL
Negative (vehicle) control = Dimethyl sulfoxide
Summarized Survival Results of Assay 2
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Relative Survival (%) |
Total number of colonies |
Cloning Efficiency (CE) |
Relative Survival (%) on plates |
+ |
5 |
A2 |
450 μg/mL |
23.2 |
187 |
0.156 |
15 |
400 μg/mL |
35.9 |
818 |
0.682 |
67 |
|||
350 μg/mL |
35.8 |
809 |
0.674 |
66 |
|||
300 μg/mL |
50.4 |
998 |
0.832 |
82 |
|||
150 μg/mL |
75.3 |
1065 |
0.888 |
87 |
|||
75 μg/mL |
86.6 |
1176 |
0.980 |
96 |
|||
37.5 μg/mL |
95.1 |
1276 |
1.063 |
105 |
|||
Negative control |
100.0 |
1221 |
1.018 |
100 |
|||
Untreated control |
103.1 |
1220 |
1.017 |
100 |
|||
Positive control (DMBA) |
23.4 |
67 |
0.056 |
5 |
|||
- |
24 |
A2 |
450 μg/mL |
0.1 |
24 |
0.020 |
2 |
400 μg/mL |
7.2 |
292 |
0.243 |
23 |
|||
350 μg/mL |
44.7 |
1193 |
0.994 |
95 |
|||
300 μg/mL |
48.5 |
1190 |
0.992 |
95 |
|||
150 μg/mL |
60.3 |
1295 |
1.079 |
104 |
|||
75 μg/mL |
85.0 |
1176 |
0.980 |
94 |
|||
37.5 μg/mL |
94.7 |
1390 |
1.158 |
111 |
|||
Negative control |
100.0 |
1250 |
1.042 |
100 |
|||
Untreated control |
94.9 |
1281 |
1.068 |
102 |
|||
Positive control (EMS) |
68.0 |
393 |
0.327 |
31 |
A2 = Assay 2
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 μg/mL
- = in the absence of S9-mix EMS = Ethyl methanesulfonate, 0.4 μL/mL
Negative (vehicle) control = Dimethyl sulfoxide
Summarized Viability Results of Assay 1
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Cloning Efficiency (CE) |
+ |
5 |
A1 |
450 μg/mL |
1066 |
0.888 |
400 μg/mL |
1115 |
0.929 |
|||
350 μg/mL |
993 |
0.828 |
|||
300 μg/mL |
1046 |
0.872 |
|||
150 μg/mL |
974 |
0.812 |
|||
75 μg/mL |
952 |
0.793 |
|||
37.5 μg/mL |
1115 |
0.929 |
|||
Negative control |
1137 |
0.948 |
|||
Untreated control |
1103 |
0.919 |
|||
Positive control (DMBA) |
933 |
0.778 |
|||
- |
5 |
A1 |
450 μg/mL |
985 |
0.821 |
400 μg/mL |
1123 |
0.936 |
|||
350 μg/mL |
915 |
0.793 |
|||
300 μg/mL |
1060 |
0.883 |
|||
150 μg/mL |
1027 |
0.856 |
|||
75 μg/mL |
1193 |
0.994 |
|||
37.5 μg/mL |
1063 |
0.886 |
|||
Negative control |
1144 |
0.853 |
|||
Untreated control |
1042 |
0.868 |
|||
Positive control (EMS) |
745 |
0.621 |
A1 = Assay 1
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 μg/mL
- = in the absence of S9-mix EMS = Ethyl methanesulfonate, 0.4 μL/mL
Negative (vehicle) control = Dimethyl sulfoxide
Summarized Viability Results of Assay 2
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Cloning Efficiency (CE) |
+ |
5 |
A2 |
450 μg/mL |
1079 |
0.899 |
400 μg/mL |
937 |
0781 |
|||
350 μg/mL |
994 |
0.828 |
|||
300 μg/mL |
1078 |
0.898 |
|||
150 μg/mL |
1074 |
0.895 |
|||
75 μg/mL |
1065 |
0.888 |
|||
37.5 μg/mL |
1005 |
0.838 |
|||
Negative control |
1107 |
0.923 |
|||
Untreated control |
910 |
0.758 |
|||
Positive control (DMBA) |
1094 |
0.912 |
|||
- |
24 |
A2 |
450 μg/mL |
581 |
0.968 |
400 μg/mL |
1088 |
0.907 |
|||
350 μg/mL |
1041 |
0.868 |
|||
300 μg/mL |
1020 |
0.850 |
|||
150 μg/mL |
999 |
0.833 |
|||
75 μg/mL |
962 |
0.802 |
|||
37.5 μg/mL |
956 |
0.797 |
|||
Negative control |
1070 |
0.892 |
|||
Untreated control |
995 |
0.829 |
|||
Positive control (EMS) |
368 |
0.307 |
A2 = Assay 2
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 μg/mL
- = in the absence of S9-mix EMS = Ethyl methanesulfonate, 0.4 μL/mL
Negative (vehicle) control = Dimethyl sulfoxide
Summarized Mutagenicity Results of Assay 1
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Mutant frequency |
+ |
5 |
A1 |
450 μg/mL |
15 |
4.2 |
400 μg/mL |
28 |
7.6 |
|||
350 μg/mL |
26 |
7.8 |
|||
300 μg/mL |
20 |
5.7 |
|||
150 μg/mL |
20 |
6.2 |
|||
75 μg/mL |
17 |
5.4 |
|||
37.5 μg/mL |
17 |
4.6 |
|||
Negative control |
27 |
7.1 |
|||
Untreated control |
19 |
5.2 |
|||
Positive control (DMBA) |
1478 |
475.6** |
|||
- |
5 |
A1 |
450 μg/mL |
20 |
6.1 |
400 μg/mL |
29 |
7.8 |
|||
350 μg/mL |
26 |
8.5** |
|||
300 μg/mL |
16 |
4.6 |
|||
150 μg/mL |
13 |
3.7 |
|||
75 μg/mL |
17 |
4.2 |
|||
37.5 μg/mL |
15 |
4.1 |
|||
Negative control |
21 |
5.5 |
|||
Untreated control |
20 |
5.7 |
|||
Positive control (EMS) |
1021 |
421.8** |
** = Statistically significant increase (at p<0.01) compared to the relevant vehicle control
A1 = Assay 1
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 μg/mL
- = in the absence of S9-mix EMS = Ethyl methanesulfonate, 0.4 μL/mL
Negative (vehicle) control = Dimethyl sulfoxide
Mutant frequencies refer to 106clonable cells.
Summarized Mutagenicity Results of Assay 2
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Mutant frequency |
+ |
5 |
A2 |
450 μg/mL |
28 |
7.7 |
400 μg/mL |
12 |
3.9 |
|||
350 μg/mL |
10 |
3.0 |
|||
300 μg/mL |
15 |
4.4 |
|||
150 μg/mL |
18 |
5.0 |
|||
75 μg/mL |
12 |
3.4 |
|||
37.5 μg/mL |
21 |
6.2 |
|||
Negative control |
14 |
3.8 |
|||
Untreated control |
15 |
5.0 |
|||
Positive control (DMBA) |
766 |
210.2** |
|||
- |
24 |
A2 |
450 μg/mL |
13 |
6.7 |
400 μg/mL |
19 |
5.3 |
|||
350 μg/mL |
32 |
9.2 |
|||
300 μg/mL |
23 |
6.8 |
|||
150 μg/mL |
20 |
6.0 |
|||
75 μg/mL |
16 |
5.0 |
|||
37.5 μg/mL |
15 |
4.7 |
|||
Negative control |
21 |
5.9 |
|||
Untreated control |
29 |
8.6 |
|||
Positive control (EMS) |
677 |
558.3** |
** = Statistically significant increase (at p<0.01) compared to the relevant vehicle control
A2 = Assay 2
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 μg/mL
- = in the absence of S9-mix EMS = Ethyl methanesulfonate, 0.4 μL/mL
Negative (vehicle) control = Dimethyl sulfoxide
Mutant frequencies refer to 106clonable cells.
Historical Control Data
(updated on 17 October 2017 using data of GLP studies)
|
Mutation frequency (Number of 6-TG resistant mutants per 106clonable cells) |
||
|
Untreated control |
||
|
5-hour, S9+ |
5-hour, S9- |
24-hour, S9- |
Mean |
18.3 |
20.7 |
19.0 |
Standard deviation |
15.1 |
16.4 |
17.2 |
Minimum |
5.1 |
5.5 |
3.3 |
Maximum |
64.1 |
55.5 |
58.0 |
n |
27 |
13 |
14 |
|
DMSO control |
||
|
5-hour, S9+ |
5-hour, S9- |
24-hour, S9- |
Mean |
21.8 |
18.9 |
18.4 |
Standard deviation |
15.9 |
11.6 |
14.4 |
Minimum |
5.4 |
6.5 |
6.8 |
Maximum |
57.3 |
47.4 |
48.5 |
n |
29 |
13 |
14 |
|
Distilled water / Water based vehicle control |
||
|
5-hour, S9+ |
5-hour, S9- |
24-hour, S9- |
Mean |
11.5 |
9.1 |
15.5 |
Standard deviation |
3.8 |
3.4 |
5.6 |
Minimum |
6.1 |
5.2 |
9.2 |
Maximum |
15.8 |
11.6 |
20.1 |
n |
6 |
3 |
3 |
|
Positive controls |
||
|
DMBA |
EMS |
EMS |
|
5-hour, S9+ |
5-hour, S9- |
24-hour, S9- |
Mean |
905.2 |
445.6 |
1176.6 |
Standard deviation |
562.7 |
118.6 |
610.9 |
Minimum |
141.2 |
239.6 |
363.1 |
Maximum |
2119.4 |
636.6 |
2449.8 |
n |
27 |
13 |
14 |
DMSO = Dimethyl sulfoxide
DMBA = 7,12-Dimethylbenz[a]anthracene
EMS = Ethyl methanesulfonate
S9+ = in the presence of S9-mix
S9- = in the absence of S9-mix
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The genetic toxicity of cis-Jasmone has been investigated in two in vitro studies. In the first study, the potential of the test substance to induce gene mutations in bacteria was tested in a bacterial gene mutation assay (Ames test) following OECD TG 471 and in compliance with GLP. The Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were tested in two independent experiments according to the plate incorporation and preincubation procedure in the absence and presence of a metabolic activation system (Phenobarbital/p-Naphthoflavone induced rat liver S9-mix). The experiments were conducted each in triplicate at concentrations from 3 to 2500 µg/plate (vehicle: DMSO). Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. It is therefore concluded that under the experimental conditions reported, Cis-Jasmone did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
The second study was an in vitro micronucleus assay in cultured peripheral human lymphocytes (with independent repeat) examining the effect of cis-Jasmone on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity was tested in two independent experiments.The study was performed according to OECD TG 487 and under GLP conditions. In the first cytogenetic assay, the test item was tested up to 440 μg/ml in the absence of S9-fraction and 495 μg/ml in the presence of S9-fraction for a 3 h exposure time with a 27 h harvest time. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 70 μg/ml for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. All positive control chemicals produced a statistically significant genotoxic effects. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. cis-Jasmone did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. Therefore, it is concluded that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
The third study is an in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of CIS-JASMONE to cause gene mutation. The study was performed according to OECD TG 476 (v. 2016) and under GLP conditions.
Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). DMSO was used as the vehicle (solvent) of the test item in this study. Treatment
concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test at up to 450 μg/mL(Assay 1, 5-hour treatment in the presence and in the absence of S9-mix), up to 450 (Assay 2, 5-hour treatment in the presence of S9-mix and 24-hour treatment in the absence of S9-mix).
In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.
In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 13.6% and 10% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 5.8% and 31% after treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. Statistically significant increase (at p<0.01 level) was observed in this experiment at 350 μg/mL concentration, although the observed value was within the general historical control range.Furthermore, the observed mutant frequencies (8.5 x 10-6) was within the expected
range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment).
In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 450 μg/mL showed a relative survival of 23.2% and 15% after treatment and on the
survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results seen in Assay 1 with metabolic activation.
In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest concentration of 450μg/mL and the following concentration 400μg/mL showed a relative survival of 0.1%, 2% and 7.2%, 23% after
treatment and on the survival plates, respectively). An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). This experiment confirmed the negative results without metabolic activation.
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. Seven evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated
concentration ranges were considered to be adequate (concentrations were tested up to the cytotoxic range in each test). The overall study was considered to be valid.
In conclusion, no mutagenic effect of CIS-JASMONE was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.
Justification for classification or non-classification
The available data provide no sufficient evidence that would imply any classification and labeling with respect to mutagenicity/genotoxicity according to Regulation (EC) No. 1272/2008 (CLP). The conclusion for classification is ‘conclusive but not sufficient for classification’.
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