Registration Dossier
Registration Dossier
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EC number: 936-831-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A bacterial reverse mutation assay according to OECD 471 and a chromosome aberration study according to OECD 473 was conducted with a structural analogue substance. The test substance (CAS 376588-17-9 was not mutagenic in bacteria and considered to be non-clastogenic.
A HPRT test in CHO cells (OECD 476) was conducted with the registered substance (EC 936-831-9). The test substance is not mutagenic in the HPRT locus assay under in vitro conditions in the absence and presence of metabolic activation.
No additional information is needed to conclude classification and labelling. For more information on the read-across approach, please refer to the Chapter 13 (attached read-across justification).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Jul 2020 - 03 Sep 2020
- 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 using the Hprt and xprt genes)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
MEDIA USED
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL) - Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 fraction was prepared according to Ames et al. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM - Test concentrations with justification for top dose:
- 0.47 μg/mL, 0.94 μg/mL, 1.88 μg/mL, 3.75 μg/mL, 7.50 μg/mL, 15.00 μg/mL, 30.00 μg/mL, 60.00 μg/mL
Following the requirements of the current international guidelines and the ICPEMC Task Group a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10-20% relative survival (adjusted cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period.
In the pre-test for toxicity based on the purity and the molecular weight of the test substance 2200.0 μg/mL (approx. 3.5 mM) was used as top concentration both with and without S9 mix at 4 hour exposure time. The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. Precipitation of the test substance in the vehicle acetone was not observed macroscopically up to the highest required concentration of 220.0 mg/mL (stock solution). In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 17.25 μg/mL and above in the absence and presence of S9 mix. Based on the data and the observations from the pre-test and taking into account the current guidelines, the following doses were selected in this study. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
Due to the insufficient solubility of the test substance in culture medium HAM`s F12, acetone was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v). - 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:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: two flasks
- Number of independent experiments : 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x1e6 cells
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 6-8 days
- Selection time (if incubation with a selective agent): 6 – 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
- If a selective agent is used: 6-thioguanine, 10 µg/ml
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: for selection of the mutants, two 175 cm² flasks with 2x1e6 cells each from every treatment group were seeded. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
Relative survival after treatment (Cloning efficiency 1 [CE1] adjusted by cell loss):
For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
Cloning efficiency 2 (CE2; viability):
For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
In all cases, after seeding the flasks or petri dishes were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.
The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity. - Evaluation criteria:
- A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit). - Statistics:
- A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment.
- Data on osmolality: Osmolality values were not relevantly influenced by test substance treatment.
CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was adversely influenced (grade > 2) in any test group tested for gene mutations.
CYTOTOXICITY
There was no decrease in the number of colonies as described by the relative survival in the presence and absence of S9 mix up to the highest evaluated concentrations for gene mutation.
STUDY RESULTS
In the absence of metabolic activation, the values for the corrected mutation frequencies (MFcorr.) ranged between 0.37 – 2.38 per 1e6 cells); the respective vehicle control had 2.78 per 1e6 cells. The obtained values were within the range of the 95% vehicle control limit (without S9 mix: MFcorr.: 0.00 – 6.21 per 1e6 cells). A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined.
In the presence of metabolic activation, the values for the corrected mutation frequencies ranged between MFcorr.: 0.35 – 3.18 per 1e6 cells; the respective vehicle control value had 1.32 per 1e6 cells. The obtained values were within the range of the 95% vehicle control limit (with S9 mix: MFcorr.: 0.00 – 7.02 per 1e6 cells. A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 237.12 per 1e6 cells; with S9 mix: MFcorr.: 73.61 per 1e6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 1e6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 1e6 cells). - Conclusions:
- Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. One experiment was carried out, in the absence and presence of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Acetone was used as vehicle. According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. The highest tested concentration (60.00 μg/mL) was based on test substance precipitation in culture medium. Test groups printed in bold type were evaluated for gene mutations:
without S9 mix 0; 0.47; 0.49; 1.88; 3.75; 7.50; 15.00; 30.00; 60.00 μg/mL
with S9 mix 0; 0.47; 0.49; 1.88; 3.75; 7.50; 15.00; 30.00; 60.00 μg/mL
Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. The highest evaluated concentration showed clear test substance precipitates in culture medium macroscopically at the end of exposure period in the presence and absence of metabolic activation. In the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
- 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:
- 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Qualifier:
- according to guideline
- Guideline:
- other: "Commission Directive 2000/32/EC, Ll 362000, Annex 4D, dated May 19, 2000
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/p-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 33; 100; 333; 1000; 2500; and 5000 µg/plate
- Vehicle / solvent:
- Ethanol (MERCK, D-64293 Darmstadt; purity > 99,8 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA 1535 and TA 100), 4-nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
Experiment I (Plate Incorporation Test):
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL Overlay agar
Experiment II (Pre-Incubation Test):
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark. Three plates were used for each dose level. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- An increase in revertant colony numbers was observed beginning at 1000 µg/plate in strain TA 1537 in the presence of metabolic activation in experiment II. This increase was judged as irrelevant since it is solely based upon a low solvent control count due to statistical fluctuations. Compared to thecorresponding negative control no increase in revertant colony numbers occurred. The data in the negative and solvent control were slightly above the historical control range in strain TA 100 (exp. I and II, with S9 mix). The number of colonies did not quite reach the lower limit of the historical control data in strain TA 1535 (exp. I, negative control with S9 mix), in strain TA 1537 (exp. II, solvent control with S9 mix) and in strain WP2 uvrA (exp. II, negative control with S9 mix). Since these deviations are rather small, these effects are considered to be based upon biologically irrelevant fluctuations in the number of colonies and have no detrimental impact on the outcome of the study.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. - Executive summary:
This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98,
and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Directive 2000/32/EC, Ll 362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 Mix
- Test concentrations with justification for top dose:
- Experiment I: 50, 100, 200, 400, 800, 1000 µg/ml with and without S9 Mix.
Experiment II: 50, 100, 200, 400, 800, 1000 µg/ml with S9 Mix and 125, 250, 500, 1000, 2000, 3000 µg/ml without S9 Mix. - Vehicle / solvent:
- ethanol (E. MERCK, D-64293 Darmstadt; purity 99.8 %). The final concentration of ethanol in the culture medium was 0.5 % (v/v). The solvent was chosen due to its solubility properties and its relative nontoxicity to the cell cultures.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol 0.5 %
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol 0.5 %
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- Treatment
Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium were added. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells. All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.
Preparation of the Cultures
15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts respectively). Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Evaluation of Cell Numbers
For evaluation of cytotoxicity indicated by reduced cell numbers additional two cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with lOOx oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation ofthe aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). - Evaluation criteria:
- A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed. - Statistics:
- Statistical significance was confirmed by means of the Fisher's exact test (10) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid: A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells) - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Toxic effects after 24 hrs continuous treatment with 3000 pg/mL in the absence of S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- PRE-TEST
No clear toxic effects were observed after 4 hrs treatment up to the highest test item concentration (concentrations: 23.4 - 3000 µg/mL). Continuous treatment for 24 hrs with 3000 µg/mL in the absence of S9 mix induced toxic effects.
In the pre-experiment, precipitation of the test item in culture medium was observed after 4 hrs treatment with 187.5 µg/mL and above in the absence of S9 mix and with 375 µg/mL and above in the presence of S9 mix. No influence of the test item on the pH value or osmolarity was observed (solvent control 398 mOsm, pH 7.3 versus 362 mOsm and pH 7.2 at 3000 µg/mL).
MAIN TEST
In the cytogenetic experiments, precipitation of the test item in culture medium was observed in experiment I at preparation interval 18 hrs with 400 µg/mL and above in the absence and the presence of S9 mix. In experiment II, in the absence of S9 mix precipitation occurred after treatment with 500 µg/mL and above at preparation interval 18 hrs, and with 250 µg/mL and above at preparation interval 28 hrs. In the presence of S9 mix, at the 28 hrs preparation interval precipitation was observed after 4 hrs treatment with 200 µg/mL and above. In this study, neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest applied concentrations of the test item. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
The test substance is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations. - Executive summary:
The test item was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The highest applied concentration in the pre-test on toxicity (3000 µg/mL; approx. 4.7 mM) was
chosen with regard to the viscosity of the test item solution using an appropriate solvent with respect to the current OECD Guideline 473. Since no relevant toxicity was observed after 4 hrs treatment in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473 in the experimental parts with 4 hrs
treatment. No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Furthermore, the highest concentrations scored for chromosomal aberrations were far in the range where test item precipitates occurred. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro and is therefore considered to be non-clastogenic in this chromosome aberration test with and without S9 mix.
Referenceopen allclose all
SUMMARY OF RESULTS
| Cytotoxicity*** | |||||||
| Exp | Exposure period [h] |
Test groups [μg/mL] |
S9 mix |
Prec.* | Genotoxicity** MFcorr. [per 106cells] |
RS [%] | CE2 [%] |
| 1 | 4 | Vehicle control1 | - | n.d. | 2.78 | 100.0 | 100.0 |
| 0.47 | - | - | n.c. | 163.3 | n.c. | ||
| 0.94 | - | - | n.c. | 139.0 | n.c. | ||
| 1.88 | - | - | 1.64 | 149.2 | 93.8 | ||
| 3.75 | - | - | 0.71 | 132.4 | 87.3 | ||
| 7.50 | - | - | 0.37 | 133.1 | 83.0 | ||
| 15.00 | - | + | 2.38 | 105.2 | 77.8 | ||
| 30.00 | - | + | n.c.1 | n.c.1 | n.c.1 | ||
| 60.00 | - | + | n.c.1 | n.c.1 | n.c.1 | ||
| Positive control2 | - | n.d | 237.12s | 106.2 | 70.7 | ||
| 1 | 4 | Vehicle control1 | + | n.d. | 1.32 | 100.0 | 100.0 |
| 0.47 | + | - | n.c. | 90.4 | n.c. | ||
| 0.94 | + | - | n.c. | 88.9 | n.c. | ||
| 1.88 | + | - | 3.18 | 84.4 | 93.1 | ||
| 3.75 | + | - | 0.74 | 123.1 | 89.5 | ||
| 7.50 | + | - | 0.35 | 97.6 | 92.8 | ||
| 15.00 | + | + | 0.43 | 92.8 | 75.7 | ||
| 30.00 | + | + | n.c.1 | n.c.1 | n.c.1 | ||
| 60.00 | + | + | n.c.1 | n.c.1 | n.c.1 | ||
| Positive control3 | + | n.d. | 73.61s | 84.4 | 71.1 | ||
* Macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle/negative control
s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)
n.c. Culture was not continued since a minimum of only four analysable concentrations is required
n.c.1 Culture was not continued since only one concentration beyond the solubility limit is required
n.d. Not determined
1 Acetone 1% (v/v)
2 EMS 400 μg/mL
3 DMBA 1.25 μg/mL
Without S9 -Mix
| Concentration µg/plate | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||||
| I | II | I | II | I | II | I | II | I | II | |
| Negative control | 12 | 13 | 17 | 6 | 51 | 35 | 183 | 165 | 52 | 32 |
| Solvent Control | 10 | 9 | 4 | 4 | 32 | 26 | 161 | 160 | 33 | 29 |
| Positive Control | 845 | 728 | 57 | 57 | 238 | 324 | 493 | 506 | 588 | 171 |
| 33 | 10 | 9 | 3 | 7 | 30 | 36 | 168 | 170 | 28 | 30 |
| 100 | 9 | 8 | 5 | 5 | 33 | 31 | 151 | 174 | 32 | 29 |
| 333 | 7 | 10 | 6 | 5 | 32 | 33 | 170 | 165 | 36 | 32 |
| 1000 | 5 | 10 | 2 | 5 | 28 | 33 | 171 | 165 | 26 | 30 |
| 2500 | 7 | 8 | 4 | 3 | 32 | 26 | 198 | 152 | 31 | 37 |
| 5000 | 5 | 6 | 4 | 4 | 28 | 33 | 164 | 173 | 19 | 33 |
I = Experiment I (Plate Incorporation Test)
II = Experiment II (Pre-Incubation Test)
Positive controls:
Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100
4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate)
methyl methane sulfonate (4.0 µL/plate) strain WP2 uvrA
With S9 -Mix
| Concentration µg/plate | TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||||
| I | II | I | II | I | II | I | II | I | II | |
| Negative control | 8 | 11 | 13 | 9 | 41 | 37 | 255 | 239 | 38 | 31 |
| Solvent Control | 11 | 10 | 8 | 5 | 31 | 38 | 247 | 222 | 39 | 32 |
| Positive Control | 203 | 178 | 70 | 140 | 510 | 1136 | 655 | 1041 | 166 | 168 |
| 33 | 10 | 8 | 5 | 6 | 33 | 38 | 211 | 221 | 46 | 31 |
| 100 | 9 | 11 | 7 | 6 | 38 | 41 | 211 | 221 | 36 | 30 |
| 333 | 8 | 7 | 7 | 7 | 36 | 32 | 243 | 228 | 33 | 32 |
| 1000 | 7 | 13 | 8 | 9 | 38 | 38 | 232 | 211 | 47 | 31 |
| 2500 | 6 | 15 | 10 | 13 | 38 | 39 | 268 | 232 | 35 | 46 |
| 5000 | 8 | 15 | 8 | 10 | 50 | 60 | 249 | 218 | 36 | 41 |
I = Experiment I (Plate Incorporation Test)
II = Experiment II (Pre-Incubation Test)
Positive controls:
2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100
2-aminoanthracene (10.0 µg/plate) strain WP2 uvrA
In both experiments, in the absence and presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells, exclusive gaps) were close to the range ofthe solvent control values (0.0 -1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.2 - 4.2 %) as compared to the rates of the solvent controls (1.9-3.5%). In both experiments, EMS (200 µg/mL) and CPA (0.7 and 1.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
According to regulation (EC) No 1907/2006, Annex XI, paragraph 1.5., substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or "category" of substances. Environmental effects or environmental fate may be predicted from data for reference substance(s) within the group by interpolation to other substances in the group (read-across approach).
The source (CAS 376588-17-9) and target (EC 936-831-9) substance share identical structural elements, the only difference being the length of the carbon chain at the ester function. The source chemical is esterified with stearic acid (C18), whereas the target compound is a mixture of stearate (C18) and palmitate (C16), with the stearate version accounting for about 60% of the chemicals present in the target compound. Therefore, 60% of the target compound are identical with the source chemical and the remaining molecules of the target compound are nearly identical with the only difference being the chain length at the ester group. It is expected that the structural difference between the octadecanoate and the hexadecanoate does not affect the physico-chemical properties, the toxicological and ecotoxicologocal profile as well as the environmental fate. Both substances are characterized by similar values for water solubility, vapor pressure and log POW. Furthermore, it can be assumed that the degradation products of CAS 376588-17-9 are very similar to the degradation products of the mixture containing the octadecanoate and the hexadecanoate. In addition, both substances triggered comparable (eco)toxicological effects. Therefore read-across to CAS 376588-17-9 is scientifically justified for physico-chemical properties, the toxicological and ecotoxicological profile as well as environmental fate. For more information on the read-across approach, please refer to the Chapter 13 (attached read-across justification).
Ames:
The mutagenic potential of CAS 376588-17-9 was investigated in an Ames test using the plate incorporation test and the pre-incubation test with Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and Escherichia coli strain WP2 uvrA both with and without liver microsomal activation. The test item was tested at the following concentrations in triplicates: 33; 100; 333; 1000; 2500; and 5000 µg/plate. No toxic effects occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Chromosome aberration:
The potential of CAS 376588-17-9 to induce structural chromosome aberrations was assessed in V79 cells in vitro in two independent experiments. In the absence of toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation. In both experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. In conclusion, the test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix
HPRT Assay:
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. One experiment was carried out, in the absence and presence of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Acetone was used as vehicle. The dose selection of this study was based on the solubility properties of the test substance in an appropriate vehicle and in culture medium in accordance to the recommendations of the current guidelines. The highest tested concentration was 60.00 μg/mL. Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. According to the results of the present in vitro study, the test substance did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were not significantly higher than the respective concurrent vehicle control values and within the 95% control limit of our historical negative control data. The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.
The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, in the absence and the presence of metabolic activation, CGX CR 946 A is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.
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