Registration Dossier
Registration Dossier
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EC number: 606-278-5 | CAS number: 19257-34-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation (bacterial reverse mutation assay / Ames test): positive in strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without metabolic activation and additionally in TA 1535 with metabolic activation [Reimann, 2000; Reimann and Görke, 2000].
Gene mutation (HPRT test): negative in V79 cells with and without metabolic activation [Chang, 2016].
32P-postlabeling study (calf thymus DNA): weak genotoxic activity [Steger-Hartmann and Naumann, 2002]
In vitro mammalian micronucleus test: OECD 487; positive (4 h with metabolic activation) [Ziegler, 2021]
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021
- 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:
- 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: V79 cells were obtained from Merck KGaA, Darmstadt
- Suitability of cells: As recommended by the OECD test guideline. The high proliferation rate were appropriate for the use of this cell line.
- Normal cell cycle time (negative control): doubling time of V79 cells in stock cultures: approximately 12 hours, determined on 03 May 2021
For cell lines:
- Absence of Mycoplasma contamination: Yes, cells were routinely checked for mycoplasma contamination
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C and 5 % CO 2 in plastic flasks. Seeding was performed with about 1 x E+05 – 5 x E+05 cells per flask.
- Cell cycle length, doubling time or proliferation index : doubling time 12 h
- Modal number of chromosomes: 22 ± 2
- Periodically checked for karyotype stability: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM (Earle’s with GlutaMAX and 25mM HEPES); Pen/Strep: 1 %; FBS: 10 %, 5% CO2, 37°C. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
-
Type and composition of metabolic activation system:
- source of S9: Sprague Dawley rats
- method of preparation of S9 mix: Liver homogenates (S9: 9000 x g fraction) were isolated in house (GLP-Prüfeinrichtung Early Development Bayer, Genetic Toxicology Wuppertal) from the livers of Aroclor 1254-induced male Sprague-Dawley rats. The used S9 fraction was derived from preparation dated 26 Nov 2019, color code green (protein content 23.8 mg/mL).
For use, frozen aliquots of the S9 fraction were slowly thawed and mixed with a cofactor solution (2+3 parts). The S9 mix contained 40 % S9 fraction to result in a final concentration of 2 % S9 in cultures and was kept in refrigerator and used on the same day. - Test concentrations with justification for top dose:
- For the test item, DMSO was selected as solvent. In this solvent the test item was soluble at
least up to 133.33 mg/mL. In the solubility test precipitation in the medium was observed at 666.7 µg/mL and above. Thus, 666.7 µg/mL was chosen for top dose. - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- vinblastine
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration sextuplicate (6 wells per concentration were measured)
- Number of independent experiments: 3:
- 4 hours treatment, 20 hours recovery, without S9 mix
- 4 hours treatment, 20 hours recovery, with S9 mix
- 24 hours treatment, without S9 mix
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2500 cells per well
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h and 24 h
- Harvest time after the end of treatment (sampling/recovery times): Approximately 24 hours after the start of treatment cells were harvested and then stained with EMA (Dye A).
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative cytotoxic effects of the test item were assessed using the relative increase in nuclei count (RINC) in the presence and absence of S9 mix. The results of the solvent controls were set 100 % and compared to the test substance treated cultures. A change of the RINC relative to the corresponding solvent control was calculated as follows:
Relative Cytotoxicity % = 100% - RINC %
METHODS FOR MEASUREMENTS OF GENOTOXICITY
The percentage of micronuclei per nucleated events (%MN), indicative of clastogenic effects,
or hypodiploid nuclei per nucleated events (%HD), indicative of aneugenic effects, was
determined. In parallel, the proportion of nuclei stemming from apoptotic or necrotic cells
was detected (%A/N).
- %A/N = (A/N / Total Events) x100
- %MN = (MN / Nucleated) x100
- %HD = (HD / Nucleated) x100
Additionally, the number of nuclei originating from viable cells was related to an internal
standard (Cell Sorting Set-up Beads) as a measure of relative increase in nuclei count. For this relative increase in nuclei count, nuclei in cultures of up to 30 parallel wells were counted at the start of treatment time to determine start values.
The percentage was calculated as follows:
Mean (Nuclei/Beads) well 1-n Test Item - Mean (Nuclei/Beads) well 1-n Start
%RINC = ---------------------------------------------------------------------------------------------------------------------------- x 100
Mean (Nuclei/Beads) well 1-n SC- Mean (Nuclei/Beads) well 1-n Start - Rationale for test conditions:
- As recommended by the OECD test guideline
- Evaluation criteria:
- Providing that all acceptability criteria were fulfilled , the test item was considered to be positive if:
- the test item induced a micronucleus frequency in one of the test item concentrations that is two-fold higher compared to the micronucleus frequency of concurrent solvent control
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data - Statistics:
- please refer to 'Any other information on materials and methods incl. tables'
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- not determined
- Remarks:
- 4h treatment. Due to an induction of the micronucleus frequency in the presence of S9 mix, micronuclei in V79 cells treated with the test item in the absence of S9 mix were neither further evaluated nor statistically analyzed.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 24.7 µg/mL
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- starting at 74.1 µg/mL after 4h treatment
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 74.1 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- STUDY RESULTS
- Concurrent vehicle negative and positive control data: see 'any other information on results incl. tables'
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o When cytokinesis block is not used: For relative cytotoxicity, additional nuclei were counted at the start of treatment. The start value was determined by calculating the nuclei beads ratio from cultures of up to 30 parallel wells resulting in a mean value of 0.1. This value was allocated for RINC which was the base of the relative cytotoxicity.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see 'any other information on results incl. tables'
- Negative (solvent/vehicle) historical control data: see 'any other information on results incl. tables' - Conclusions:
- The present study was conducted according to OECD guideline 487 (2016). Chinese hamster lung fibroblasts (V79) were exposed to 0, 0.3, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, and 666.7 µg/mL Dimethoxyketal for 4h (with and without metabolic acitvation) and 24 h (without metabolic activation). The frequency of micronuclei, apoptotic/necrotic nuclei and of hypodiploid nuclei were determined. Under the experimental conditions reported the test item induced chromosome breakage (structural chromosomal aberrations) leading to micronucleus formation stemming from V79 cells in vitro in the presence of metabolic activation.
- Executive summary:
In a mammalian cell micronucleus assay according to OECD guideline 487 (2016), V79 cells cultured in vitro were exposed to Dimethoxyketal in DMSO at concentrations of 0, 0.3, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, and 666.7 µg/mL in the presence and absence of mammalian metabolic activation [rat S9 liver mix] for either 4 h (with and without metabolic activation) and 24 h (without metabolic activation).
Dimethoxyketal was tested up to cytotoxic concentrations (i.e., 24.7 µg/mL (24 h) and 74.1 µg/mL (4 h. With S9 mix, the test item showed a statistically significant increase in the number of micronuclei at a concentration of 74.1 μg/mL. The induction of the micronucleus frequency was less than two-fold, but the increase was considered biologically relevant since it was outside the range of the historical solvent control. The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline 487 for in vitro mammalian cell micronucleus data.
Based on the described results Dimethoxyketal is considered to increase micronuclei in Chinese hamster lung fibroblasts (V79) after metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Mar 2000
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 May 1983
- Deviations:
- no
- Principles of method if other than guideline:
- The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine gene locus
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced male rat liver S9 mix
- Test concentrations with justification for top dose:
- 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- cyclophosphamide
- other: 4-nitro-o-phenylenediamine (10 µg; only TA 1537), 2-nitrofluorene (only TA 1538 and 98), sodium azide (only TA 1535 and 100), benzo(a)pyrene (only TA 98 and 100), cyclophosphamide (only TA 1535), 2-aminoanthracene (2.5 µg; all strains)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: between 12 and 29.8 x E+08 cells/mL
- Test substance added in medium; in agar (plate incorporation)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically. - Evaluation criteria:
- The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was consic:fered if the number of revertants of the compound groups compared to the number of revertants of the negative group was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: not data
- Precipitation and time of the determination: Precipitates in the agar were found at 5.0 mg/plate.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: For the determination of the base-line mutation rates 0.05 mL phosphate buffer or DMSO were employed as negative or solvent control, respectively. In order to check the activity of the metabolizing system and the mutability of the bacteria at least two control mutagens were tested for each strain. Their mutagenic effect occurred either directly (4-NPDA, 2-NF, NaN3) or after metabolic activation (2-AA, BP, CP). The colony counts recorded on appropriate negative control plates confirmed the characteristically spontaneous reversion rates of the tester strains. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains. Precipitates in the agar were found at 5.0 mg/plate. Growth inhibition of the background lawn was not observed.
Ames test:
- Signs of toxicity: no. Growth inhibition of the background lawn was not observed.
- Individual plate counts:
TA1535: 216, 213, 258
TA1537: 141, 199, 188
TA1538: 132, 117, 171
TA100: 274, 311, 309
TA98: 113, 114, 134
- Mean number of revertant colonies per plate and standard deviation:
Please refer to table 2 under any other information on results incl. tables. - Conclusions:
- The mutagenic potential of Dimethoxyketal was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was seen with the tester strain TA 1535 in the absence and presence of S9 mix as well as with the tester strains TA 98, TA 100, TA 1537 and TA 1538 in the absence of S9 mix. Therefore, the test substance was considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Dimethoxyketal in DMSO at concentrations of 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.
The test item was tested up to the limit concentration of 5000 µg/plate. Precipitates were found in the agar at 5000 µg/plate. Tester strains TA1535 and TA98 showed increased dose-dependent reversion to prototrophy in assays with the test item at the doses tested between 0.5 and 5.0 mg/plate, in the absence and presence (TA1535) or only in the absence of S9 mix (TA98). TA1537 and TA1538 showed a positive response only at 2.5 mg/plate without S9 mix. Due to the dose-dependency the response of TA100 is also considered positive in the assay without S9 mix, although in no case was the number of revertants doubled in comparison to the spontaneous rate. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed.
This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
The test material is considered mutagenic under the conditions of the test.
Based on the positive test results of this test, i.e. without the use of the tester strains S. typhimurium TA102 or E.coli WP2 uvr, a repetition of the test including the lacking tester strains is considered dispensible.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June to July 2000
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 May 1983
- Deviations:
- yes
- Remarks:
- only pre-incubation procedure was performed
- Principles of method if other than guideline:
- The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine gene locus
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced male rat liver S9 mix
- Test concentrations with justification for top dose:
- 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- cyclophosphamide
- Remarks:
- 4-nitro-o-phenylenediamine (10 µg; without metabolic activation; only TA 1537), 2-aminoanthracene (5.0 µg; with metabolic acitvation; all strains); N-nitrosodimethylamine (5 µL; only TA 100)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments. One
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Between 7.5 and 37.9 X E+08 cell/mL
- Test substance added in medium; preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically. - Evaluation criteria:
- The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if the number of revertants of the compound groups compared to the number of. revertants of the negative group was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 250 µg/plate onwards
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 250 µg/plate onwards
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 500 µg/plate onwards
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 250 µg/plate onwards
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Precipitation and time of the determination: Precipitates in the agar were found at the highest dose tested in the tests without and with S9mix.
STUDY RESULTS
- Concurrent vehicle negative and positive control data:
For the determination of the base-line mutation rates 0.05 mL phosphate buffer or DMSO were employed as negative or solvent control, respectively. In order to check the activity of the metabolizing system and the mutability of the bacteria at least two control mutagens were tested for each strain. Their mutagenic effect occurred either directly (4-NPDA, 2-NF, NaN3) or after metabolic activation (2-AA, BP, CP, DMNA). Sterility controls were performed additionally.
Ames test:
- Signs of toxicity: Growth inhibition of the background lawn was observed in the tests without S9 mix from 0.5 mglplate (TA 1535) or from 0.25 mg/plate (TA100, TA 1537, TA 1538) onwards and at the highest dose of 5.0 mg/plate (TA98). In the tests with S9 mix growth inhibition was only found at the highest dose tested at the five tester strains.
- Individual plate counts:
TA1535: 284, 226, 255
TA1537: 66, 72, 87
TA1538: 223, 257, 253
TA100: 224, 329, 354
TA98: 416, 427, 293
- Mean number of revertant colonies per plate and standard deviation
Please refer to table 2 under any other information on results incl. tables. - Conclusions:
- The mutagenic potential of the test substance was evaluated in the modified Ames test (pre-incubation method) with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was seen with all tester strains up to 5000 µg/plate in the absence of S9 mix. However, no mutagenic response was observed in the experiments with metabolic activation (S9 mix). Therefore, the test substance was considered to be mutagenic in the Ames Salmonella/microsome test using the pre-incubation modification.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Dimethoxyketal in DMSO at concentrations of 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.
The test item was tested up to the limit concentration of 5000 µg/plate. All of the five tester strains TA1535, TA100, TA1537, TA1538 and TA98 showed increased reversion to prototrophy in assays with the test item in the absence of S9 mix, whereby TA1535 and TA98 were the most sensitive strains.
In the present study the test item was tested up to the highest recommended dose of 5.0 mg/plate. Precipitates in the agar were found at the highest dose tested in the tests without and with S9 mix.
Growth inhibition of the background lawn was observed in the tests without S9 mix from 0.5 mg/plate (TA1535) or from 0.25 mg/plate (TA100, TA1537, TA1538) onwards and at the highest dose of 5.0 mg/plate (TA98). In the tests with S9 mix growth inhibition was only found at the highest dose tested at the five tester strains. The positive controls induced the appropriate responses in the corresponding strains.
This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
The test material is considered mutagenic under the conditions of the test.
Based on the positive test results of this test, i.e. without the use of the tester strains S. typhimurium TA102 or E.coli WP2 uvr, a repetition of the test including the lacking tester strains is considered dispensible.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Aug to Nov 2016
- 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:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM with supplements; for the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: S9 mix from the liver of phenobarbital/ß-naphthoflavone induced rats
- source of S9: rat liver
- method of preparation of S9 mix: Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Test facilities SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl 2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). - Test concentrations with justification for top dose:
- Pre-experiment: 3.3, 8.2, 20.5, 51.2, 128, 320, 800, 2000 µg/mL (-/+ S9 mix)
Main experiment: 6.5, 13, 26, 39, 52 µg/mL (-/+ S9 mix) - Vehicle / solvent:
- Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- THF
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: ethylmethanesulfonate (EMS; 2.4 mM), 7,12-dimethylbenzanthracene (DMBA; 8.9 µM)
- Remarks:
- EMS was used without and DMBA with metabolic activation
- Details on test system and experimental conditions:
- - DOSE SELECTION: According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye and microscopically. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 3.3 μg/mL and 2000 μg/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476. In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed from 51.2 to 800 µg/mL without metabolic activation and at 51.2 µg/mL and above with metabolic activation. The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Precipitation occurred after 4 hours treatment at 320 µg/mL and above with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were generally spaced by a factor of 2.0. Narrower spacing was used at high concentrations to cover the toxic range more closely. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations. The cultures at the two lowest concentration with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.
- CULTURE MEDIUM: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- SEEDING: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×107 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- TREATMENT: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G". Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium. Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below. Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II). The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: one
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.7 - 1.2 E+07 cell/flask
- Test substance added in medium
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 3 to 4 days until second subcultivation and 7 days after second subcultivation until incubation with selection medium.
- Selection time (if incubation with a selective agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5E+05 cells each in medium containing 6-TG 11 µg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II). The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation and time of the determination: Precipitation occurred after 4 hours treatment at 320.0 µg/mL and above with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES (if applicable): A pre-test was performed in order to determine the toxicity of the test item.
The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment. In addition the pH-value and the osmolarity were measured.
In the pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is evident as a reduction of the cloning efficiency (CE). The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 3.3 µg/mL and 2000 µg/mL were used. In the pre-experiment strong cytotoxic effects, indicated by a relative cloning efficiency of 50% or below were observed from 51.2 to 800.0 µg/mL without metabolic activation and at 51.2 µg/mL and above with metabolic activation. The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were generally spaced by a factor of 2.0.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: In the main experiment with and without S9 mix the range of the solvent controls was from 18.1 up to 41.0 mutants per 1 x E+06 cells; the range of the groups treated with the test item was from 11.6 up to 52.3 mutants per 1 x E+06 cells. The highest solvent control value of 41.0 mutants per 1 x E06 cells exceeded the 95% confidence interval but the mutation frequency of the parallel culture and the mean value of both cultures (18.1 and 41.0 equal to a mean of 29.6) was fully acceptable. The viability (cloning efficiency II) of the solvent control in the second culture of the main experiment with and without metabolic activation did not quite reach the lower limit of 50%. The data are valid however, as the solvent controls of the parallel culture exceeded this limit. EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred at 52.0 mg/mL with and without metabolic activation. The recommended toxic range of approximately 10% - 20% rel. adjusted cloning efficiency I was covered with and without metabolic activation.
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 2 x E+06 cells/175cm² flask (subcultivation one; treatment); 500 cells/25 cm² flask for relative survival. 2 x E+06 cells/ 175 cm² flask (subcultivation two; treatment).
o Number of cells plated in selective and non-selective medium: 4 to 5 x E+05 cells/75 cm² flask (selection). 500 cells/ 25 cm² flask (viability/cloning efficiency II). - Conclusions:
- The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 52 µg/mL with and without metabolic activation. Relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred at 52.0 µg/mL with and without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance was considered to be non-mutagenic in this HPRT assay.
- Executive summary:
In a mammalian cell gene mutation assay in V79 cells [HPRT] according to OECD 476, V79 Chinese hamster lung fibroblast cells cultured in vitro were exposed to Dimethoxyketal in Tetrahydrofuran at concentrations of 6.5, 13, 26, 39, 52 µg/mL in the presence and absence of mammalian metabolic activation [S9 mix from the liver of phenobarbital/ß-naphthoflavone induced rats].
The test item was tested up to cytotoxic concentrations, i.e. no relevant cytotoxic effect was observed from 51.2 to 800 µg/mL without metabolic activation and at 51.2 µg/mL and above with metabolic activation. Precipitation occurred after 4 hours treatment at 320 µg/mL and above with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 18.1 up to 41.0 mutants per 106 cells; the range of the groups treated with the test item was from 11.6 up to 52.3 mutants per 106 cells. The highest solvent control value of 41.0 mutants per 106 cells exceeded the 95% confidence interval but the mutation frequency of the parallel culture and the mean value of both cultures (18.1 and 41.0 equal to a mean of 29.6) was fully acceptable. The positive controls EMS (300 μg/mL) and DMBA (2.3 μg/mL) showed a distinct increase in induced mutant colonies. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix.
This study is classified as acceptable or. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity data.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- July to Oct 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- The test substance was incubated with 100 µg calf thymus DNA at concentrations of 20, 100, 250 and 500 µg/mL corresponding to 63 µmol/L, 314 µmol/L, 785 µmol/L and 1.6 mmol/L with two replicates each. For this purpose a stock solution of the test substances was prepared in DMSO and added to the DNA in aqueous solution to 1% (v/v). As a negative control calf thymus DNA without test compound was incubated in 1% DMSO in two replicates. Substances with known direct DNA reactivity such as tetrachloro-p-benzoquinone (p-chloranile, 246 µg/L = 1 mmol/L and 492 µg/mL = 2 mmol/L) and sulfoxymethylpyrene (0.17 µg/mL = 0.5 µmol/L) dissolved in DMSO served as positive controls.
After incubation for 2 h at 37°C the DNA was extracted from the incubation mixture with phenol/chloroform and precipitated with ethanol. The DNA was redissolved in 10 mmol/L Tris buffer (pH 8,0) and kept frozen at about -75°C until further preparation.
The adducts were enriched by nuclease P1 treatment. The subsequent DNA-adduct analysis was performed by means of the 32P-postlabeling technique and the labeled adducts were separated by thin layer chromatography. DNA from rats treated in vivo with cyproterone acetate (CPA) was used as reference standard for labeling efficiency and chromatographic resolution.
The preparation of the samples up to a subsequent DNA-adduct analysis 'isolation and hydrolysis of DNA, adduct enrichment, labeling of the nucleotides and thin layer chromatography (TLC)' were performed according to internal SOPs. - GLP compliance:
- not specified
- Type of assay:
- other: 32P-postlabeling
- Target gene:
- calf thymus DNA
- Test concentrations with justification for top dose:
- 63, 314, 785, 1600 µmol/L
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: p-chloranile, sulfoxymethylpyrene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: one
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: The test item was incubated with 100 µg calf thymus DNA at concentrations of 20, 100, 250 and 500 µg/mL corresponding to 63 µmol/L, 314 µmol/L, 785 µmol/L and 1.6 mmol/L with two replicates each. For this purpose a stock solution of the test substances was prepared in DMSO and added to the DNA in aqueous solution to 1% (v/v). As a negative control calf thymus DNA without test compound was incubated in 1% DMSO in two replicates. Substances with known direct DNA reactivity such as tetrachloro-p-benzoquinone (p-chloranile, 246 µg/L = 1 mmol/L and 492 µg/mL = 2 mmol/L) and sulfooxymethylpyrene (0.17 µg/mL = 0.5 µmol/L) dissolved in DMSO.
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: for 2 h at 37°C
- OTHER: After incubation for 2 h at 37°C the DNA was extracted from the incubation mixture with phenol/chloroform and precipitated with ethanol. The DNA was redissolved in 10 mmol/L Tris buffer (pH 8,0) and kept frozen at about -75°C until further preparation. The adducts were enriched by nuclease P1 treatment. The subsequent DNA-adduct analysis was performed by means of the 32 P-postlabeling technique and the labeled adducts were separated by thin layer chromatography. DNA from rats treated in vivo with cyproterone acetate (CPA) was used as reference standard for labeling efficiency and chromatographic resolution.
The preparation of the samples up to a subsequent DNA-adduct analysis isolation and hydrolysis of DNA, adduct enrichment, labeling of the nucleotides and thin layer
chromatography (TLC) were performed. - Evaluation criteria:
- After incubation for 2 h at 37°C the DNA was extracted from the incubation mixture with phenol/chloroform and precipitated with ethanol. The DNA was redissolved in 10 mmol/L Tris buffer (pH 8,0) and kept frozen at about -75°C until further preparation. The adducts were enriched by nuclease P1 treatment. The subsequent DNA-adduct analysis was performed by means of the 32P-postlabeling technique and the labeled adducts were separated by thin layer chromatography. DNA from rats treated in vivo with cyproterone acetate (CPA) was used as reference standard for labeling efficiency and chromatographic resolution.
- Key result
- Species / strain:
- other: calf thymus DNA
- Metabolic activation:
- not specified
- Genotoxicity:
- other: weak genotoxic activity
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- In a 32P-postlabeling study on the adduct forming potential the test substance displayed a weak genotoxic activity. Since no enzymes were existent in the chosen cell-free test system (calf thymus DNA) the observed genotoxicity was caused by a direct reactivity of the test substance to DNA. The reactivity may be most probably attributed to the ketal moiety in position 3 of the steroid.
- Executive summary:
In a mammalian 32P-postlabeling study the adduct forming potential of the test substance was investigated. The test substance was incubated with 100 µg calf thymus DNA at concentrations of 20, 100, 250 and 500 µg/mL corresponding to 63 µmol/L, 314 µmol/L, 785 µmol/L and 1.6 mmol/L with two replicates each. As a negative control calf thymus DNA without test compound was incubated in 1% DMSO in two replicates. Substances with known direct DNA reactivity such as tetrachloro-p-benzoquinone (p-chloranile, 246 µg/L = 1 mmol/L and 492 µg/mL = 2 mmol/L) and sulfoxymethylpyrene (0.17 µg/mL = 0.5 µmol/L) dissolved in DMSO served as positive controls. After incubation for 2 h at 37°C the DNA was extracted from the incubation mixture with phenol/chloroform and precipitated with ethanol. The adducts were enriched by nuclease P1 treatment. The subsequent DNA-adduct analysis was performed by means of the 32P-postlabeling technique and the labeled adducts were separated by thin layer chromatography. DNA from rats treated in vivo with cyproterone acetate (CPA) was used as reference standard for labeling efficiency and chromatographic resolution.
Thin layer chromatography runs with the postlabeled samples from the incubations with 314 µmol/L, 785 µmol/L and 1.600 µmol/L of the test item showed one weak but reproducible spot at an exposure time of 16 hours which was not detectable in the negative control samples. No spot was detected at the lowest tested concentration of 63 µmol/L.
The positive control p-chloranile led to an adduct pattern with two distinct spots at 2 mmol/L. At 1 mmol/L this pattern was barely discernible. The positive control sulfoxymethylpyrene (0.5 µmol/L) led to a distinct adduct pattern already after 15 min exposure time. The CPA reference DNA showed its typical diagonal adduct pattern as known from previous experiments and literature reports together with some additional spots of unknown origin.
It can therefore be concluded that the test item displayed a weak genotoxic activity. Since no enzymes were existent in the chosen cell-free test system the observed genotoxicity was caused by a direct reactivity of the test item to DNA. The reactivity may be most probably attributed to the ketal moiety in position 3 of the steroid.
This study is classified as acceptable as supporting information for the information requirements of Regulation (EU) No. 1907/2006 (REACH) Annex VII section 8.4.1.
Referenceopen allclose all
Frequency of Micronuclei
With S9 mix, the test item showed a statistically significant increase in the number of micronuclei at a concentration of 74.1 μg/mL. The induction of the micronucleus frequency was less than two-fold, but the increase was considered biologically relevant since it was outside the range of the historical solvent control.
Moreover, a concentration-related trend in the micronucleus frequency across the increasing concentration levels of the test item was found following 4 hours treatment with S9 mix.
Thus, overall, the micronucleus test showed a relevant increase in the micronucleus frequency in V79 cells treated with the test item in the presence of S9 mix (4 hours treatment). Due to an induction of the micronucleus frequency in the presence of S9 mix, micronuclei in V79 cells treated with the test item in the absence of S9 mix were neither further evaluated nor statistically analyzed.
Frequency of Apoptotic/Necrotic Nuclei
No biologically relevant increases in the numbers of apoptotic/necrotic nuclei were detected after 4 hours treatment. After the 24 h treatment period an increase in the number of apoptotic/necrotic nuclei (16 % A/N) was detected at a test item concentration of 74.1 µg/mL. This concentration was excluded from analysis due to excessive cytotoxicity.
Frequency of Hypodiploid Nuclei
No biologically relevant increases of numbers of hypodiploid nuclei were detected after 4 hours treatment. The same was true for a treatment period of 24 hours.
Results and concurrent control data:
Historical Controls
9000 – 18000 nuclei per study on flow cytometer MACSQuant 10 or Accuri C6 were evaluated.
Historical Controls 2018-2020, 4 Hours Treatment, 24 Hours Harvest Time | |||||||
| Micronuclei in % | ||||||
Solvent or Substance | S9 Mix | Conc. | Number of studies | Mean | SD | Min | Max |
Water | - | 1% v/v | 11 | 1.1 | 0.6 | 0.4 | 2.5 |
DMSO | - | 1% v/v | 150 | 1.0 | 0.4 | 0.4 | 2.0 |
Mitomycin C | - | 0.1 µg/mL | 168 | 15.8 | 3.7 | 7.0 | 27.4 |
Water | + | 1% v/v | 11 | 1.4 | 0.5 | 0.7 | 2.4 |
DMSO | + | 1% v/v | 162 | 1.2 | 0.4 | 0.5 | 2.2 |
CP | + | 2 µg/mL | 173 | 16.0 | 4.3 | 5.9 | 29.2 |
Historical Controls 2018 - 2020, 24 Hours Treatment, 24 Hours Harvest Time | |||||||
Water | - | 1% v/v | 12 | 1.2 | 0.8 | 0.4 | 2.9 |
DMSO | - | 1% v/v | 158 | 1.2 | 0.5 | 0.4 | 2.5 |
Vinblastine | - | 0.0018 µg/mL | 177 | 18.1 | 5.6 | 8.5 | 42.6 |
Summary of the Results (4 Hours Treatment –S9 Mix) | ||||||
| Conc. µg/mL | % A/N | %MN | %HD | % rel. Cytotoxicity | Precipitation |
Solvent control | 0.0 | 0.8 | 1.1 | 0.1 | - | no |
Positive control MMC | 0.1 | 2.8 | 18.2# | 0.3 | 39.3 a | no |
Test item | 0.3 | 0.7 | 1.1 | 0.1 | 0.0 | no |
| 0.91 | 0.8 | 1.2 | 0.1 | 0.0 | no |
| 2.74 | 0.8 | 1.2 | 0.1 | 0.0 | no |
| 8.23 | 0.7 | 1.0 | 0.1 | 0.0 | no |
| 24.7 | 0.9 | 1.4 | 0.1 | 28.0 a | no |
| 74.1 | 2.9 | 2.6 | 0.1 | 96.3 b | no |
| 222.2 | DIV/0! | DIV/0! | DIV/0! | DIV/0! c | no |
| 666.7 | DIV/0! | DIV/0! | DIV/0! | DIV/0! c | yes |
a relevant cytotoxicity b excessive cytotoxicity, (above limit = 55 ± 5%) c concentration excluded from flow cytometric assessment # biologically relevant increase | ||||||
| ||||||
Summary of the Results (4 Hours Treatment +S9 Mix) | ||||||
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| Conc. µg/mL | % A/N | %MN | %HD | % rel. Cytotoxicity | Precipitation |
Solvent control | 0.0 | 0.7 | 1.3 | 0.1 | - | no |
Positive control CP | 2 | 3.2 | 15.5* | 0.1 | 34.5 a | no |
Test item | 0.3 | 0.5 | 1.3 | 0.1 | 13.0 | no |
| 0.91 | 0.7 | 1.4 | 0.1 | 2.7 | no |
| 2.74 | 0.8 | 1.4 | 0.1 | 0.0 | no |
| 8.23 | 1.0 | 1.3 | 0.1 | 0.2 | no |
| 24.7 | 0.7 | 1.6 | 0.1 | 9.2 | no |
| 74.1 | 1.0 | 2.4* | 0.1 | 45.2 a | no |
| 222.2 | DIV/0! | DIV/0! | DIV/0! | DIV/0! c | no |
| 666.7 | DIV/0! | DIV/0! | DIV/0! | DIV/0! c | yes |
a relevant cytotoxicity c concentration excluded from flow cytometric assessment * statistically significant increase of micronucleated events (P = < 0.05) | ||||||
| ||||||
Summary of the Results (24 Hours Treatment –S9 Mix) | ||||||
| Conc. µg/mL | % A/N | %MN | %HD | % rel. Cytotoxicity | Precipitation |
Solvent control | 0.0 | 0.6 | 1.3 | 0.1 | - | no |
Positive control VSS | 0.00018 | 2.9 | 20.8# | 5.5# | 65.5 b | no |
Test item | 0.3 | 0.4 | 1.2 | 0.1 | 0.9 | no |
| 0.91 | 0.5 | 1.3 | 0.1 | 0.0 | no |
| 2.74 | 0.5 | 1.3 | 0.1 | 0.0 | no |
| 8.23 | 0.5 | 1.1 | 0.1 | 11.8 | no |
| 24.7 | 1.3 | 1.0 | 0.1 | 79.0 b | no |
| 74.1 | 16.4 | 1.8 | 0.3 | 106.4 b | no |
| 222.2 | DIV/0! | DIV/0! | DIV/0! | DIV/0! c | no |
| 666.7 | DIV/0! | DIV/0! | DIV/0! | DIV/0! c | yes |
b excessive cytotoxicity, (above limit = 55 ± 5%) c concentration excluded from flow cytometric assessment # biologically relevant increase | ||||||
The total colony counts of the 1E+06 dilution of bacterial suspension culture confirmed the viability and high cell density of the cultures of the individual strains.The colony counts recorded on appropriate negative control plates confirmed the characteristically spontaneous reversion rates of the tester strains. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.
Tester strains TA1535 and TA98 showed increased dose-dependent reversion to prototrophy in assays with the test item at the doses tested between 0.5 and 5.0 mg/plate, in the absence and presence (TA1535) or only in the absence of S9 mix (TA98). TA1537 and TA1538 showed a positive response only at 2.5 mg/plate without S9 mix. Due to the dose-dependency the response of TA100 is also considered positive in the assay without S9 mix, although in no case was the number of revertants doubled in comparison to the spontaneous rate.
In the present study the test item was tested up to the highest recommended dose of 5 mg/plate. Precipitates in the agar were found at 5.0 mg/plate. Growth inhibition of the background lawn was not observed.
Table1:
| TA1535 | TA100 | TA1537 | TA1538 | TA98 | |||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
DMSO | 14±1 | 9±5 | 107±16 | 96±21 | 19±8 | 16±3 | 13±6 | 19±4 | 41±6 | 32±9 |
phosphate buffer | 14±2 | 15±3 | 116±11 | 101±14 | 22±2 | 17±3 | 11±2 | 22±4 | 44±5 | 45±8 |
2-AA | 15±1 | 145±4 | 144±4 | 1515±15 | 10±3 | 7±2 | 20±10 | 538±51 | 48±7 | 677±41 |
CP | 47±5 | 236±8 |
|
|
|
|
|
|
|
|
BaP 2.5 µg |
|
| 100±12 | 985±89 |
|
|
|
| 40±4 | 301±60 |
4-NPDA |
|
|
|
| 96±19 | 20±3 |
|
|
|
|
2-NF |
|
|
|
|
|
| 1239±62 | 555±39 | 959±34 | 414±36 |
NaN3 | 676±24 | 185±35 | 744±39 | 217±10 |
|
|
|
|
|
|
Table 2:
| TA1535 | TA100 | TA1537 | TA1538 | TA98 | |||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
DMSO | 14±1 | 9±5 | 107±16 | 96±21 | 19±8 | 16±3 | 13±6 | 19±4 | 41±6 | 32±9 |
phosphate buffer | 14±2 | 15±3 | 116±11 | 101±14 | 22±2 | 17±3 | 11±2 | 22±4 | 44±5 | 45±8 |
0.025 mg | 16±5 | 10±1 | 102±16 | 93±9 | 18±6 | 17±3 | 8±3 | 23±2 | 43±6 | 48±5 |
0.05 mg | 13±3 | 13±2 | 112±9 | 92±8 | 17±5 | 13±3 | 12±1 | 20±5 | 46±2 | 42±4 |
0.10 mg | 16±4 | 8±1 | 102±6 | 90±9 | 21±9 | 16±4 | 9±3 | 22±5 | 63±6 | 40±4 |
0.25 mg | 21±3 | 13±1 | 124±9 | 94±20 | 22± | 14±3 | 10±3 | 24±5 | 70±11 | 41±4 |
0.50 mg | 28±7 | 12±7 | 132±14 | 100±15 | 13±3 | 19±6 | 16±1 | 19±1 | 100±9 | 46±4 |
1.00 mg | 40±10 | 20±2 | 165±19 | 91±13 | 16±2 | 15±7 | 14±4 | 22±3 | 191±13 | 42±11 |
2.50 mg | 85±20 | 29±4 | 175±28 | 47±4 | 48±2 | 13±2 | 31±9 | 21±6 | 222±31 | 47±5 |
5.00 mg | 59±5 | 16±2 | 127±23 | 47±6 | 10±3 | 7±2 | 19±6 | 16±4 | 155±12 | 35±3 |
The total colony counts of the 1E+06 dilution of bacterial culture confirmed the viability and high cell density of the cultures of the individual strains. The colony counts recorded on appropriate negative control plates confirmed the characteristically spontaneous reversion rates of the tester strains. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.
All of the five tester strains TA1535, TA100, TA1537, TA1538 and TA98 showed increased reversion to prototrophy in assays with the test item in the absence of S9 mix, whereby TA1535 and TA98 were the most sensitive strains.
In the present study the test item was tested up to the highest recommended dose of 5.0 mg/plate. Precipitates in the agar were found at the highest dose tested in the tests without and with S9 mix.
Growth inhibition of the background lawn was observed in the tests without S9 mix from 0.5 mg/plate (TA1535) or from 0.25 mg/plate (TA100, TA1537, TA1538) onwards and at the highest dose of 5.0 mg/plate (TA98). In the tests with S9 mix growth inhibition was only found at the highest dose tested at the five tester strains.
Table 1:
| TA1535 | TA100 | TA1537 | TA1538 | TA98 | |||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
DMSO | 12±4 | 15±3 | 133±3 | 90±7 | 4±1 | 11±2 | 12±2 | 22±2 | 34±5 | 43±13 |
phosphate buffer | 14±6 | 16±3 | 142±16 | 91±8 | 4±2 | 16±3 | 10±1 | 26±0 | 38±7 | 42±3 |
2-AA | 6±1 | 88±11 | 229±10 | 401±58 | 7±3 | 46±4 | 10±4 | 287±27 | 25±5 | 293±34 |
CP | 60±7 | 561±24 |
|
|
|
|
|
|
|
|
DMNA |
|
| 131±15 | 1137±186 |
|
|
|
|
|
|
BaP 2.5 µg |
|
|
|
|
|
|
|
| 33±3 | 394±9 |
4-NPDA |
|
|
|
| 86±8 | 25±6 |
|
|
|
|
2-NF |
|
|
|
|
|
| 1263±17 | 337±32 | 975±25 | 280±25 |
NaN3 | 666±15 | 50±7 | 852±12 | 97±2 |
|
|
|
|
|
|
Table 2:
| TA1535 | TA100 | TA1537 | TA1538 | TA98 | |||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
DMSO | 12±4 | 15±3 | 133±3 | 90±7 | 4±1 | 11±2 | 12±2 | 22±2 | 34±5 | 43±13 |
phosphate buffer | 14±6 | 16±3 | 142±16 | 91±8 | 4±2 | 16±3 | 10±1 | 26±0 | 38±7 | 42±3 |
0.025 mg | 17±4 | 15±4 | 124±19 | 88±5 | 7±4 | 13±4 | 9±3 | 25±3 | 26±7 | 46±4 |
0.05 mg | 16±5 | 14±5 | 121±13 | 84±7 | 4±0 | 14±5 | 10±2 | 24±9 | 34±7 | 42±6 |
0.10 mg | 15±5 | 16±3 | 125±6 | 95±12 | 7±4 | 15±1 | 10±3 | 21±5 | 48±4 | 46±1 |
0.25 mg | 21±0 | 16±3 | 147±54 | 93±7 | 8±2 | 10±2 | 4±1 | 20±4 | 75±15 | 39±6 |
0.50 mg | 35±1 | 15±4 | 115±130 | 84±9 | 9±5 | 10±3 | 3±3 | 20±2 | 103±4 | 44±1 |
1.00 mg | 43±4 | 16±3 | 351±57 | 67±1 | 6±5 | 8±3 | 26±15 | 15±6 | 146±5 | 51±2 |
2.50 mg | 42±6 | 9±3 | 276±37 | 54±4 | 9±8 | 5±2 | 30±5 | 19±2 | 169±8 | 43±8 |
5.00 mg | 36±8 | 12±6 | 308±28 | 60±8 | 14±9 | 7±1 | 18±6 | 17±2 | 185±5 | 46±7 |
The main experiment was evaluated at the following concentrations: 6.5, 13, 26, 39 and 52 µg/mL (-/+ S9 mix).
Relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred at 52.0 mg/mL with and without metabolic activation. The recommended toxic range of approximately 10% - 20% rel. adjusted cloning efficiency I was covered with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.
The 95% confidence interval was exceeded at several experimental points. However, the test run at any one of the experimental points exceeding the 95% confidence interval was not significant at all occasions with a single exception. A significant t-test was noted at an intermediate concentration of 26.0 μg/mL with metabolic activation. This result however, was judged as biologically irrelevant as it was not reproduced at any other, even higher concentration and there was no dose-dependent increase as indicated by the non-significant trend test.
In the main experiment with and without S9 mix the range of the solvent controls was from 18.1 up to 41.0 mutants per 106 cells; the range of the groups treated with the test item was from 11.6 up to 52.3 mutants per 106 cells. The highest solvent control value of 41.0 mutants per 106 cells exceeded the 95% confidence interval but the mutation frequency of the parallel culture and the mean value of both cultures (18.1 and 41.0 equal to a mean of 29.6) was fully acceptable.
The viability (cloning efficiency II) of the solvent control in the second culture of the main experiment with and without metabolic activation did not quite reach the lower limit of 50%. The data are valid however, as the solvent controls of the parallel culture exceeded this limit.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Thin layer chromatography runs with the postlabeled samples from the incubations with 314 µmol/L, 785 µmol/L and 1.600 µmol/L of the test item showed one weak but reproducible spot at an exposure time of 16 hours which was not detectable in the negative control samples. No spot was detected at the lowest tested concentration of 63 µmol/L.
The positive control p-chloranile led to an adduct pattern with two distinct spots at 2 mmol/L. At 1 mmol/L this pattern was barely discernible. The positive control sulfoxymethylpyrene (0.5 µmol/L) led to a distinct adduct pattern already after 15 min exposure time. The CPA reference DNA showed its typical diagonal adduct pattern as known from previous experiments and literature reports together with some additional spots of unknown origin.
It can therefore be concluded that the test item displayed a weak genotoxic activity. Since no enzymes were existent in the chosen cell-free test system the observed genotoxicity was caused by a direct reactivity of the test item to DNA. The reactivity may be most probably attributed to the ketal moiety in position 3 of the steroid.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In a mammalian cell gene mutation assay in V79 cells [HPRT] according to OECD 476, V79 Chinese hamster lung fibroblast cells cultured in vitro were exposed to Dimethoxyketal in Tetrahydrofuran at concentrations of 6.5, 13, 26, 39, 52 µg/mL in the presence and absence of mammalian metabolic activation [S9 mix from the liver of phenobarbital/ß-naphthoflavone induced rats].
The test item was tested up to cytotoxic concentrations, i.e. no relevant cytotoxic effect was observed from 51.2 to 800 µg/mL without metabolic activation and at 51.2 µg/mL and above with metabolic activation. Precipitation occurred after 4 hours treatment at 320 µg/mL and above with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 18.1 up to 41.0 mutants per 106 cells; the range of the groups treated with the test item was from 11.6 up to 52.3 mutants per 106 cells. The highest solvent control value of 41.0 mutants per 106 cells exceeded the 95% confidence interval but the mutation frequency of the parallel culture and the mean value of both cultures (18.1 and 41.0 equal to a mean of 29.6) was fully acceptable.]. The positive controls EMS (300 μg/mL) and DMBA (2.3 μg/mL) showed a distinct increase in induced mutant colonies. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity data.
In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Dimethoxyketal in DMSO at concentrations of 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.
The test item was tested up to the limit concentration of 5000 µg/plate. Precipitates were found in the agar at 5000 µg/plate. Tester strains TA1535 and TA98 showed increased dose-dependent reversion to prototrophy in assays with the test item at the doses tested between 0.5 and 5.0 mg/plate, in the absence and presence (TA1535) or only in the absence of S9 mix (TA98). TA1537 and TA1538 showed a positive response only at 2.5 mg/plate without S9 mix. Due to the dose-dependency the response of TA100 is also considered positive in the assay without S9 mix, although in no case was the number of revertants doubled in comparison to the spontaneous rate. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed.
This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
The test material is considered mutagenic under the conditions of the test.
In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Dimethoxyketal in DMSO at concentrations of 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.
The test item was tested up to the limit concentration of 5000 µg/plate. All of the five tester strains TA1535, TA100, TA1537, TA1538 and TA98 showed increased reversion to prototrophy in assays with the test item in the absence of S9 mix, whereby TA1535 and TA98 were the most sensitive strains.
In the present study the test item was tested up to the highest recommended dose of 5.0 mg/plate. Precipitates in the agar were found at the highest dose tested in the tests without and with S9 mix.
Growth inhibition of the background lawn was observed in the tests without S9 mix from 0.5 mg/plate (TA1535) or from 0.25 mg/plate (TA100, TA1537, TA1538) onwards and at the highest dose of 5.0 mg/plate (TA98). In the tests with S9 mix growth inhibition was only found at the highest dose tested at the five tester strains. The positive controls induced the appropriate responses in the corresponding strains.
This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
The test material is considered mutagenic under the conditions of the test.
In a mammalian 32P-postlabeling study the adduct forming potential of the test substance was investigated. The test substance was incubated with 100 µg calf thymus DNA at concentrations of 20, 100, 250 and 500 µg/mL corresponding to 63 µmol/L, 314 µmol/L, 785 µmol/L and 1.6 mmol/L with two replicates each. As a negative control calf thymus DNA without test compound was incubated in 1% DMSO in two replicates. Substances with known direct DNA reactivity such as tetrachloro-p-benzoquinone (p-chloranile, 246 µg/L = 1 mmol/L and 492 µg/mL = 2 mmol/L) and sulfoxymethylpyrene (0.17 µg/mL = 0.5 µmol/L) dissolved in DMSO served as positive controls. After incubation for 2 h at 37°C the DNA was extracted from the incubation mixture with phenol/chloroform and precipitated with ethanol. The adducts were enriched by nuclease P1 treatment. The subsequent DNA-adduct analysis was performed by means of the 32P-postlabeling technique and the labeled adducts were separated by thin layer chromatography. DNA from rats treated in vivo with cyproterone acetate (CPA) was used as reference standard for labeling efficiency and chromatographic resolution.
Thin layer chromatography runs with the postlabeled samples from the incubations with 314 µmol/L, 785 µmol/L and 1.600 µmol/L of the test item showed one weak but reproducible spot at an exposure time of 16 hours which was not detectable in the negative control samples. No spot was detected at the lowest tested concentration of 63 µmol/L.
The positive control p-chloranile led to an adduct pattern with two distinct spots at 2 mmol/L. At 1 mmol/L this pattern was barely discernible. The positive control sulfoxymethylpyrene (0.5 µmol/L) led to a distinct adduct pattern already after 15 min exposure time. The CPA reference DNA showed its typical diagonal adduct pattern as known from previous experiments and literature reports together with some additional spots of unknown origin.
It can therefore be concluded that the test item displayed a weak genotoxic activity. Since no enzymes were existent in the chosen cell-free test system the observed genotoxicity was caused by a direct reactivity of the test item to DNA. The reactivity may be most probably attributed to the ketal moiety in position 3 of the steroid.
This study is classified as acceptable as supporting information for the information requirements of Regulation (EU) No. 1907/2006 (REACH) Annex VII section 8.4.1.
In a mammalian cell micronucleus assay according to OECD guideline 487 (2016), V79 cells cultured in vitro were exposed to Dimethoxyketal in DMSO at concentrations of 0, 0.3, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, and 666.7 µg/mL in the presence and absence of mammalian metabolic activation [rat S9 liver mix] for either 4 h (with and without metabolic activation) and 24 h (without metabolic activation).
Dimethoxyketal was tested up to cytotoxic concentrations (i.e., 24.7 µg/mL (24 h) and 74.1 µg/mL (4 h. With S9 mix, the test item showed a statistically significant increase in the number of micronuclei at a concentration of 74.1 μg/mL. The induction of the micronucleus frequency was less than two-fold, but the increase was considered biologically relevant since it was outside the range of the historical solvent control. The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline 487 for in vitro mammalian cell micronucleus data.
Based on the described results Dimethoxyketal is considered to increase micronuclei in Chinese hamster lung fibroblasts (V79) after metabolic activation.
Due to the equivocal results obtained from the studies summarized above a mutagenic potential for the substance is suspected.
Justification for classification or non-classification
- Two reverse gene mutation assays in bacteria were performed according to OECD TG 471, one using the plate incorporation method and one using the pre-incubation method. In the test using the plate incorporation method no cytotoxicity was observed and the number of revertants were increased but not doubled in comparison to the spontaneous rate. In the test using the pre-incubation method precipitates were found at the highest dose level and reduction of the background lawn was seen from 0.5 mg/plate or 0.25 mg/plate onwards.
- In a supporting study the adduct forming potential was studied with calf thymus DNA. Since this method is a cell-free method the direct interaction with the DNA was measured. Effects on cells could not be determined with this method. The results showed that Dimethoxyketal has only a weak genotoxic potential in the presence of DNA. It is expected that this potential is due to the ketal moiety in position 3 of the steroid.
- In a study conducted according to OECD TG 476, the genotoxic potential to mammalian cells was investigated. There was no substantial increase of the mutation frequency above that of the solvent controls for the test item.
- However, in a study conducted according to OECD guideline 487 Dimethoxyketal increased the frequency of micronuclei in chinese hamster lung fibroblasts (V79) after 4h (with metabolic activation). Although this increase was not twofold as compared to the negative control, the values were outside the historical data range for the negative control and, thus, the result is considered to be biologically relevant.
In summary, Dimethoxyketal exhibited genotoxic properties in bacteria (Ames), in mammalian cells (V79; MNT in vitro) and on pure DNA (32P-postlabeling assay), however, was negative in a mutagenicity test with mammalian cells (HPRT). A direct reactivity to DNA might be attributed to the ketal moiety in position 3 of the steroid.
Based on these equivocal results a mutagenic potential can be suspected, thus a self-classification as germ cell mutagen Cat. 2 according to Regulation (EU) No. 1272/2008 (CLP) is justified.
According to the current REACH mutagenicity testing strategy following positive results in a gene mutation test in bacteria and a micronucleus test in vitro a subsequent in vivo testing shall be considered (e.g. a combined in vivo Comet Assay (OECD TG 489) and Micronucleus Test (OECD TG 474)), however is omitted since the need to conduct the assay should be weighed against animal-welfare considerations and it is not expected that it will change safety measures for workers.
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