Registration Dossier
Registration Dossier
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EC number: 230-237-7 | CAS number: 6976-37-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation (reference 7.6.1-1).
The genotoxicity of the substance was investigated in an in vitro micronucleus test according to OECD test guideline 487 under GLP-conditions. The substance was considered as not genotoxic under the test conditions (reference 7.6.1-2).
The mutagenicity of the substance was investigated in an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD test guideline 476 under GLP-conditions. Under the experimental conditions of this study the test item did not induce a positive result in the HPRT test. Therefore, the test item is considered to be "non-mutagenic under the conditions of the HPRT assay" (reference 7.6.1-3).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-08-29 to 2016-09-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine and tryptophan
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Remarks:
- E.coli WP2uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- method of preparation of S9 mix : The complete S9 Mix was freshly prepared. with components: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL + Rat liver homogenate (S9) 100 mL + Salt solution for S9 Mix 400 mL
- volume of S9 mix in the final culture medium: 0.5 mL of the S9 Mix was added to each overlay tube. - Test concentrations with justification for top dose:
- First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances.
- Vehicle / solvent:
- - Vehicle used: ultrapure water for test item, SAZ and MMS; DMSO for NPD, 9AA and 2AA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-1,2-phenylenediamine (NPD, TA98 without metabolic activation) and 2-aminoanthracene (2AA, all strains, with metabolic activation)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) and preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 ºC
- Exposure duration/duration of treatment: ca. 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; revertant colony numbers - Evaluation criteria:
- A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- 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:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of the test item stock solutions (50 mg/mL) were 9.39 and 9.18. The pH of the different overlays without test item stock solution was in the range of 7.12 - 7.30, and the pH of overlays completed with test item stock solution was in the range of 7.44 - 7.50.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Precipitation and time of the determination: No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).
RANGE-FINDING STUDY: Based on the solubility test, revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate of the test item. In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
Ames test:
- Signs of toxicity : No cytotoxicity was detected.
- Please refer to "Any other information on results" for details.
HISTORICAL CONTROL DATA: Please refer to table 3. - Conclusions:
- In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation.
- Executive summary:
The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.
Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:
5000; 1600; 500; 160; 50 and 16 μg/plate.
In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 9.24. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solution (50 mg/mL) were 9.39 and 9.18. The pH of the test item containing overlays was in the range of 7.44 - 7.50 in both experiments. In this study the alkaline test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-07-31 to 2017-11-10
- 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:
- adopted 29. July 2016
- Deviations:
- yes
- Remarks:
- Please refer to "Principles of method if other than guideline"
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 30. May 2008
- Deviations:
- yes
- Remarks:
- Please refer to "Principles of method if other than guideline"
- Principles of method if other than guideline:
- In experiment IIa the value of the solvent control of the positive control was outside the range of the 95 % control limit. This is uncritical since nevertheless, the positive control showed a statistically significant increase in comparison to this solvent control. In addition a second experiment II was performed for verification of the results of the first experiment II.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: V79 cell line, from CLS (Eppelheim, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years be-cause of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a high cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Normal cell cycle time (negative control): doubling time 12 - 16 h in stock cultures, no exact value given but checked with every experiment and data is stored in laboratory archive
For cell lines:
- Absence of Mycoplasma contamination: yes, stocks were screened
- Number of passages: not specified
- Methods for maintenance in cell culture: cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
DMEM, Supplier Biochrom AG, 12247 Berlin, Germany, serving as base for:
1. Complete Culture medium (DMEM, 5 % Horse Serum, 1 % Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O)
2. Selection medium (DMEM, 5 % Horse Serum, 1 % Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O, 2 µg/mL 6-Thioguanine (1 mg/mL))
37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 - 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: Trinova Biochem GmbH, Gießen, Germany, batch no. 3691; produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
- method of preparation of S9 mix: 10 mL Phosphate buffer for S9-Mix + 1.25 mL NADP-solution + 0.175 mL Glu-6-phosphate-solution + 0.6 mL Salt-solution for S9-Mix + 3 mL S9
- concentration or volume of S9 mix and S9 in the final culture medium: final concentration of the S9 fraction during treatment was 0.4 %
- quality controls of S9: not specified - Test concentrations with justification for top dose:
- concentration in experiment I and II: 0.06, 0.13, 0.25, 0.5, 1, 2 mg/mL
A pre-test was performed to determine the doses for the main experiments. In the pre-test 0.03, 0.06, 0.13, 0.25, 0.5, 1 and 2 mg/mL were used. The highest dose was selected based on the OECD guideline and the pre-test. - Vehicle / solvent:
- - Solvents used: DMSO for positive control 7,12-dimethylbenz(a)anthracene (DMBA); DMEM without supplements for the test item and the positive control Ethylmethane sulfonate (EMS)
- Justification for choice of solvent: DMEM was chosen as solvent, because this vehicle has no effects on the viability of cells at the used concentration, does not show genetic toxicity and the test item was completely soluble at the required concentration (20 mg/mL). - 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: duplicate
- Number of independent experiments: 2 (1 experiment I, 2 experiments II)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding:
1E6 cells per 10 cm culture dish and 500 cells (for the determination of the cytotoxicity) per 6 cm culture dish
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
24 h
- Exposure duration/duration of treatment:
experiment I: 4 h (with and without S9); experiments II: 24 h (without S9)
- Harvest time after the end of treatment:
for determination of the cytotoxicity (6 cm dished): 8 day-incubation time in experiment I and 7 day-incubation time in experiments II; for viability and mutagenicity: 168 h + 8 day-incubation time in experiment I and 7 day-incubation time in experiments II
FOR GENE MUTATION:
- Expression time:
168 h
- Selection time:
8 day-incubation time in experiment I and 7 day-incubation time in experiments II
- Fixation time: cytotoxicity: 4 h + 8 h or 24 h + 7 d; viability and mutagenicity: 4 + 168 h + 8 d or 24 + 168 h + 7 d
- Selective agent used: 6-thioguanine TG with final concentration: 2 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells after expression time: mutagenicity: 1E5 +/- 1E4 cells per 10 cm culture dish; viability: 500 +/- 10 cells per 6 cm culture dish; cell colonies were stained (0.1 % Löffler's methylene blue Solution in 0.01 % KOH solution) and counted
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Calculation of mutant frequency - Rationale for test conditions:
- The conditions of experiment 1 are according to the recommendations of the OECD TG 476.
The second experiment was performed to confirm the findings of experiment 1. - Evaluation criteria:
- The assay is considered acceptable if:
1. the MF found in the solvent controls falls within the laboratory historical 95 % control limit.
2. the PC substances must produce a significant increase (p < 0.05) in MF and lies in the range of the laboratory historical 95 % control limit.
3. 2 experimental conditions (+S9 and -S9) are tested unless one resulted in positive results.
4. adequate number of cells (spontaneous MF is 5 - 20 per 1E6 cells) and concentrations (minimum of 4) are analysable.
5. the criteria for the selection of top concentration are in line with guideline requirements.
Providing that all acceptability criteria are fulfilled:
a) test chemical is considered clearly positive if, in any of the experimental conditions examined:
• at least one of the concentrations exhibits a statistically significant increase compared with the concurrent NC,
• the increase is concentration-related when evaluated with an appropriate trend test,
• any of the results are outside the distribution of the historical NC data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
b) test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent NC,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical NC data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data.
When the response is not clearly negative or clearly positive, or to assist in establishing the biological relevance of a result, the data should be evaluated by expert and/or further investigations. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A p-value of 0.05 or lower (significance level 95 %) was considered as significant.
Statistical significance at the 1 % (p < 0.01) resp. 5 % level (p <0.05) was evaluated by means of chi-square-test. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The osmolality and the pH values of the solvent controls, the positive controls as well as the test item concentrations (in DMEM medium with 5 % horse serum (HS)) that were used in the pre-test were determined to exclude a negative influence on the assay by those parameters. None of the tested positive controls or test item concentrations provoked a critical change of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Possibility of evaporation from medium: Not specified, but not expected due to vapour pressure of test item.
- Water solubility: Not applicable. Test item was sufficiently soluble in medium.
- Precipitation and time of the determination: No precipitation detected.
- Definition of acceptable cells for analysis: Not specified
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: A pre-test was performed to determine the test concentrations for the main experiments.
STUDY RESULTS
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Please refer to attached background material.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Please refer to attached background material. - Conclusions:
- Under the experimental conditions of this study the test item did not induce a positive result in the HPRT test. Therefore, the test item is considered to be "non-mutagenic under the conditions of the HPRT assay".
- Executive summary:
A study according to OECD 476 was performed to investigate the potential of the test item to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X Chinese Hamster V79 cells.
The assay comprised a pre-test and three independent experiments (experiment I and two experiments II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.
The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The two second experiments (experiment IIa and IIb) were performed with a treatment period of 24 hours without metabolic activation.
The following nominal concentrations of the test item were investigated: 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL and 0.06 mg/mL.
The highest nominal concentration (2 mg/mL) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Precipitation of the test item was not observed up to the highest tested concentration.
EMS (experiment I: 300 µg/mL, both experiments II: 150 µg/mL) and DMBA (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies but without reduction of the viability. Thus, they demonstrated enough sensitivity of the testing procedure and the activity of the metabolic activation system.
No cytotoxic effects were observed in any of the test item concentrations in experiment I. Therefore, all concentrations were analyzable for mutagenicity.
In the approach with metabolic activation (experiment I) no dose dependant effect was observed. The values of the solvent and positive control remained well within the range of the historical data. At the highest test item concentration (2 mg/mL), no significant increase in mutant frequency was detected. However, at the 2 next lower test item concentrations (1 mg/mL and 0.5 mg/mL) a discrepancy between the results of replicate A and B was noticed. At the concentration 1 mg/mL a significant increase in mutant frequency was observed at replicate A. At the concentration 0.5 mg/mL the mutant frequency of replicate B induced a significantly increased value. At the test item concentrations 0.25 mg/mL and 0.13 mg/mL no considerable effect was observed. At the lowest test item concentration a significant effect was detected in replicate A but not in replicate B.
A similar effect was observed in the approach without metabolic activation. Again the values of the solvent and positive control remained well within the range of the historical data but for example at the highest test item concentration (2 mg/mL) replicate B induced a strong increase in mutant colonies whereas no increase was detected in replicate A. At the next lower test item concentration (1 mg/mL) the mutant frequency was significantly increased in both replicates. At the next lower test item concentration (0.5 mg/mL) no significant increase was detected. However, at the test item concentration 0.25 mg/mL and 0.13 mg/mL, again the values fluctuate between low mutant frequencies as well as significantly increased mutant frequencies. At the lowest test item concentration both replicates induced a statistically significant increase in mutant frequency.
In experiment IIa the viability was reduced but still within the non-cytotoxic range. Therefore in this experiment the four highest test item concentrations were evaluated. In the highest test item concentration the mutant frequency was significantly increased in replicate B whereas the value of replicate A remained within the historical data. At the next lower test item concentration no effect was detected. At the two lower test item concentrations (0.5 mg/mL and 0.25 mg/mL) one replicate respectively induced a significant in-crease in mutant frequency but the second replicate did not induce such an effect.
In those experiments (experiment I and IIa) no dose-dependent effect was observed. In addition the differences between the concentrations and replicates indicate that there is no obvious mutagenic effect.
Nevertheless, because of the large differences between the single replicates, especially in experiment IIa, a second experiment II (experiment IIb) was performed, to verify the results of experiment IIa and to determine their biologically relevance.
In experiment IIb no cytotoxic effects were observed in any of the test item concentrations. No statistically significant increase in mutant colony numbers/10^6 cells was observed up to the maximum concentration in replicate A, B as well as in the mean values. All values remained well within the historical control range and a dose-dependent effect was not detected. Therefore, the result of experiment IIb is clearly negative.
In accordance with the OECD 476, in experiment I (-S9) and IIa the criteria for a clearly positive or a clearly negative result are not fulfilled. In experiment I (+S9) as well as in experiment IIb the result is clearly negative.
Since the equivocal results in the approach without metabolic activation in experiment I and IIa could not be confirmed in a third experiment (experiment IIb), they are declared as biologically not relevant. Therefore, the overall result of this HPRT test is considered negative.
In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce a positive result in the HPRT test. Therefore, the result of the HPRT test with the test item is considered to be "non-mutagenic under the conditions of the HPRT assay".
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-05-22 to 2017-08-09
- 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:
- adopted 29. July 2016
- Deviations:
- yes
- Remarks:
- Please refer to "Principles of method if other than guideline".
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.49: "In Vitro Mammalian Cell Micronucleus Test"
- Version / remarks:
- adopted 14. Feb. 2017
- Deviations:
- yes
- Remarks:
- Please refer to "Principles of method if other than guideline".
- Principles of method if other than guideline:
- Details on deviation from guideline:
Determination of cytotoxicity in experiment II: Only 499 cells were evaluated in the solvent control of the positive control (NaCl 0.9 %). The deviation is uncritical since it is only mar-ginal and has no significant effect on the end result of the positive control. A consequence on the end result of the test item can be excluded. - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: primary human lymphocytes from young (approximately 18 - 35 years of age) non¬smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chem¬icals, ionizing radiations) at levels that would increase the background incidence of micro-nucleate cells
- Suitability of cells: Primary cultures of human peripheral lymphocytes are preferred for this type of study be-cause of their low and stable background rate of micronuclei. In addition, human cells are generally the most relevant ones for risk assessment.
- Normal cell cycle time (negative control): not specified
For lymphocytes:
- Sex, age and number of blood donors: male: 31 years, female: 26 years (donor sample was excluded (experiment set as invalid) due to extremely high number of binucleated cells containing micronuclei (1.95 %) in solvent control)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: yes, phytohaemagglutinin
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: RPMI 1640 (from Biochrom AG) wad used as a basis for complete culture medium (15 % FCS, 1 % Penicillin/Streptomycin, 2 % phytohaemagglutinin solution) and minimal culture medium (MCM) (1 % Penicillin/Streptomycin, 2 % phytohaemagglutinin solution). The cell cul-tures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH, Gießen, Germany, batch no. 3653; produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
- method of preparation of S9 mix: 10 mL Phosphate buffer for S9-Mix + 1.25 mL NADP-solution +0.175 mL Glu-6-phosphate-solution + 0.6 mL Salt-solution for S9-Mix + 3 mL S9
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL S9 mix per mL medium
- quality controls of S9: not specified - Test concentrations with justification for top dose:
- - test concentrations (in the experiment): 0.13, 0.25, 0.5, 1, 2 mg/mL
- concentrations scored for micronuclei: 0.5, 1, 2 mg/mL
- On the basis of the cytokinesis-block proliferation index the concentrations were selected for micronuclei scoring. - Vehicle / solvent:
- - Solvents used: minimal culture medium for test item; 0.9 % NaCl for positive controls
- Justification for choice of solvent: MCM was chosen as solvent, because this solvent has no effects on the viability of cells, does not show genetic toxicity and the test item was completely soluble. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 72 h for initial growth, then preparation of duplicates with direct seeding with treatment
- Exposure duration/duration of treatment: experiment 1: 4 h; experiment 2: 23.5 h
- Harvest time after the end of treatment: directly after treatment for experiment 2 (23.5 h); for experiment 1 washing after 4 h treatment and afterwards additional growth for 19 h with cytoB (23 h)
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: colchicine, in experiment 2, 0.03 and 0.035 µg/mL
- Cytokinesis blocked method: cytokinesis blocking substance: cytoB, at 5 µg/mL, experiment 2 23.5 h, experiment 1 19 h
- Methods of slide preparation and staining technique used including the stain used: The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10 % solution of Giemsa. All slides were independently coded before microscopic analysis.
- Number of cells spread and analysed per concentration: At least 1000 binucleated cells per culture were scored for micronuclei. However, for cytotoxicity in experiment II only 499 cells were evaluated in the solvent control of the positive control (NaCl 0.9 %).
- Criteria for scoring micronucleated cells: Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index - Rationale for test conditions:
- According to recommendation in the OECD TG 487 for lymphocytes, i.e. for the initial experiment 3-6h and for the subsequent experiment 1.5-2 cell cycles (18-24h)
- Evaluation criteria:
- Comparison of binucleated cells with micronuclei of each of the treatment groups with the solvent control group.
Acceptability
The genotoxicity assay is considered acceptable if it meets all of the following criteria:
• All experimental conditions are tested (short exposure with and without metabolic activation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
• In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.
• The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
• The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.
• The criteria for cell proliferation and for the selection of concentrations are fulfilled.
Classification
The test item is considered to have no genotoxic effects if it meets all of the following crite-ria:
• Neither a statistically significant nor a concentration-related increase of the number of micronucleated cells in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for solvent controls.
The test item is considered to have genotoxic effects if, in any of the experimental conditions, it meets all of the following criteria:
• At least one test concentration shows a statistically significant increase of micronucle-ated cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleated cells can be observed.
• Any of the results lies outside the range of the historical laboratory control data for solvent controls.
When all of these criteria are met, the test chemical is considered to be able to induce chromosomal breaks and/or gain or loss of genetic material in this test system. - Statistics:
- Fisher's exact test with a significance level of 5 % (for each treatment-control comparison).
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- with and without metabolic activation
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: None of the tested controls or the test item provoked a critical change of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Possibility of evaporation from medium: Not specified, but not expected due to vapour pressure of test item.
- Water solubility: Not applicable. Test item was sufficiently soluble in medium.
- Precipitation and time of the determination: No precipitation detected.
- Definition of acceptable cells for analysis: Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis.
STUDY RESULTS
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: Please refer to attached background material.
- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells: Please refer to attached background material.
HISTORICAL CONTROL DATA
Please refer to attached background material. - Conclusions:
- The test item does not induce the formation of micronuclei in human lymphocytes in vitro.
- Executive summary:
A study according to OECD 487 was performed to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor
The test item was dissolved in minimal culture medium to prepare a stock solution (20 mg/mL), corresponding to the highest concentration (2 mg/mL) in the test. In addition, a geometric series of dilutions was prepared from the stock solution.
Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. Cytotoxicity and level of micronuclei were determined. After the culture harvest time, the cells were harvested and slides were prepared. Then, the proportion of cells containing micronuclei was determined.
Two valid independent experiments (experiment I and experiment II) were performed. In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with medium control, solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected.
No cytotoxic effect was detected at any of the tested concentrations in both experiments.
Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed.
All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.
In conclusion, under the experimental conditions reported, the test item does not induce the formation of micronuclei in human lymphocytes in vitro.Therefore, the test item is considered as "not genotoxic" under the conditions of the test.
Referenceopen allclose all
Table 1: Summary Table of the Results of the Initial Mutation Test (Plate Incorporation Test)
| Concentrations (µg/plate) |
Salmonella typhimurium tester strains | Escherichia coli | ||||||||||||||||||
| TA 98 | TA100 | TA 1535 | TA 1537 | WP2 uvrA | ||||||||||||||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | |||||||||||
| Mean values of revertants per plate Mutation rate (MR) | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR |
| Untreated Control | 17 | 0.98 | 20.3 | 0.94 | 88.7 | 0.81 | 144.7 | 1.17 | 9 | 0.96 | 11.7 | 1.09 | 8.7 | 1.63 | 5.3 | 1 | 22.3 | 1.24 | 24.3 | 1 |
| DMSO Control | 17.3 | 1 | 22.3 | 1 | - | - | 109.7 | 1 | - | - | 9 | 1 | 7.7 | 1 | 7.7 | 1 | - | - | 25.3 | 1 |
| Ultrapure Water Control | 17.3 | 1 | 21.7 | 1 | 110 | 1 | 123.7 | 1 | 9.3 | 1 | 10.7 | 1 | 5.3 | 1 | 5.3 | 1 | 18 | 1 | 24.3 | 1 |
| 5000 | 17.7 | 1.02 | 22 | 1.02 | 98.7 | 0.9 | 126 | 1.02 | 12.7 | 1.36 | 14 | 1.31 | 5 | 0.94 | 4.7 | 0.88 | 19.3 | 1.07 | 31 | 1.27 |
| 1600 | 21 | 1.21 | 27.3 | 1.26 | 95.3 | 0.87 | 122 | 0.99 | 13 | 1.39 | 12.8 | 1.19 | 4.7 | 0.88 | 8 | 1.5 | 16.3 | 0.91 | 26 | 1.07 |
| 500 | 17.3 | 1 | 21.3 | 0.98 | 107.3 | 0.98 | 120.7 | 0.98 | 14.3 | 1.54 | 13 | 1.22 | 7 | 1.31 | 5.3 | 1 | 20 | 1.11 | 30.7 | 1.26 |
| 160 | 14.7 | 0.85 | 26 | 1.2 | 117.3 | 1.07 | 130.7 | 1.06 | 12.7 | 1.36 | 13 | 1.22 | 5 | 0.94 | 6.7 | 1.25 | 23.3 | 1.3 | 31.7 | 1.3 |
| 50 | 17.7 | 1.02 | 21 | 0.97 | 106.7 | 0.97 | 136 | 1.1 | 11 | 1.18 | 10 | 0.94 | 4.7 | 0.88 | 7.3 | 1.38 | 16.7 | 0.93 | 23.3 | 0.96 |
| 16 | 18 | 1.04 | 26.7 | 1.23 | 100.3 | 0.91 | 136 | 1.1 | 8.7 | 0.93 | 9.3 | 0.88 | 4.3 | 0.81 | 5 | 0.94 | 20.3 | 1.13 | 32 | 1.32 |
| NPD (4 µg/plate) | 470 | 27.12 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| SAZ (2 µg/plate) | - | - | - | - | 1549.3 | 14.08 | - | - | 979.3 | 104.93 | - | - | - | - | - | - | - | - | - | - |
| 9AA (50 µg/plate) | - | - | - | - | - | - | - | - | - | - | - | - | 860 | 112.17 | - | - | - | - | - | - |
| MMS (2 µL/plate) | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 617.3 | 34.3 | - | - |
| 2AA (2 µg/plate) | - | - | 1176 | 52.66 | - | - | 1008 | 9.19 | - | - | 179.3 | 19.93 | - | - | 169 | 22.04 | - | - | - | - |
| 2AA (50 µg/plate) | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 342 | 13.5 |
MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.
Table 2: Summary Table of the Results of the Confirmatory Mutation Test (Pre-incubation Test)
| Concentrations (µg/plate) |
Salmonella typhimurium tester strains | Escherichia coli | ||||||||||||||||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | WP2 uvrA | ||||||||||||||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | |||||||||||
| Mean values of revertauts per plate Mutation rate (MR) | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR | Mean | MR |
| Untreated Control | 15.7 | 1.09 | 20.3 | 1.02 | 74.7 | 0.86 | 115 | 0.97 | 12.3 | 1 | 9.7 | 1.04 | 7.3 | 1.47 | 6.7 | 1.17 | 19 | 1.04 | 29.7 | 0.75 |
| DMSO control | 13.3 | 1 | 14.7 | 1 | - | - | 100.3 | 1 | - | - | 10 | 1 | 5.3 | 1 | 6.3 | 1 | - | - | 36 | 1 |
| Ultrapure Water Control | 14.3 | 1 | 20 | 1 | 86.3 | 1 | 118.3 | 1 | 12.3 | 1 | 9.3 | 1 | 5 | 1 | 5..7 | 1 | 18.3 | 1 | 39.7 | 1 |
| 5000 | 19 | 1.33 | 15.7 | 0.78 | 98 | 1.14 | 99.7 | 0.84 | 10.7 | 0.78 | 10.3 | 1.11 | 8 | 1.6 | 7 | 1.24 | 25 | 1.36 | 39.7 | 1 |
| 1600 | 15.3 | 1.07 | 17..7 | 0.88 | 87 | 1.01 | 100.3 | 0.85 | 14.3 | 1.05 | 9 | 0.96 | 7 | 1.4 | 7.3 | 1.29 | 28.7 | 1.56 | 36.7 | 0.92 |
| 500 | 17.7 | 1.23 | 15.7 | 0.78 | 82 | 0.95 | 104 | 0.88 | 13 | 0.95 | 9 | 0.96 | 8 | 1.6 | 4.7 | 0.82 | 26.7 | 1.45 | 33 | 0.83 |
| 160 | 19.3 | 1.35 | 20.3 | 1.03 | 95 | 1.1 | 103.7 | 0.88 | 13 | 0.95 | 9.3 | 1 | 4 | 0.8 | 5.3 | 0.94 | 25.7 | 1.4 | 29.3 | 0.74 |
| 50 | 19.7 | 1.37 | 17.3 | 0.87 | 85.7 | 0.99 | 98.3 | 0.83 | 18.3 | 1.34 | 8 | 0.86 | 7 | 1.4 | 6 | 1.06 | 21 | 1.15 | 29.3 | 0.74 |
| 16 | 15 | 1.05 | 15.3 | 0.77 | 94 | 1.09 | 112.3 | 0.95 | 12 | 0.88 | 12.7 | 1.36 | 7 | 1.4 | 6 | 1.06 | 23 | 1.25 | 28.3 | 0.71 |
| NPD (4 µg/plate) | 293.7 | 22.03 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| SAZ (2 µg/plate) | - | - | - | - | 852 | 9.87 | - | - | 968 | 78.49 | - | - | - | - | - | - | - | - | - | - |
| 9AA (50 µg/plate) | - | - | - | - | - | - | - | - | - | - | - | - | 598.7 | 112.25 | - | - | - | - | - | - |
| MMS (2 µL/plate) | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 960 | 52.36 | - | - |
| 2AA (2 µg/plate) | - | - | 597.3 | 40.73 | - | - | 1186.7 | 11.83 | - | - | 120.7 | 12.07 | - | - | 96 | 15.16 | - | - | - | - |
| 2AA (50 µg/plate) | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 165.3 | 4.59 |
MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine, SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.
Table 3: Historical Control Values for Revertants Plate (for the Period of 2008-2015)
| Bacterial strains | |||||||
| Historical control data of untreated control | TA98 | TA100 | TA1535 | TA1537 | E. coli | ||
| Average | 21.4 | 106 | 10.4 | 8.1 | 25.6 | ||
| -S9 | SD | 3.7 | 27.3 | 1.5 | 2.5 | 2.5 | |
| Minimum | 9 | 65 | 3 | 2 | 11 | ||
| Maximum | 39 | 157 | 23 | 19 | 45 | ||
| TA98 | TA100 | TA1535 | TA1537 | E. coli | |||
| Average | 28 | 117.1 | 11.9 | 9 | 34.3 | ||
| +S9 | SD | 4.2 | 19.4 | 1.5 | 2 | 5.4 | |
| Minimum | 12 | 75 | 4 | 3 | 18 | ||
| Maximum | 48 | 166 | 24 | 20 | 56 | ||
| Bacterial strains | |||||||
| Historical control data of DMSO control | TA98 | TA100 | TA1535 | TA1537 | E. coli | ||
| Average | 20.9 | 101.4 | 10.3 | 7.9 | 24.9 | ||
| -S9 | SD | 3.5 | 26.2 | 1.4 | 2.5 | 4.9 | |
| Minimum | 10 | 65 | 3 | 2 | 11 | ||
| Maximum | 39 | 150 | 23 | 20 | 44 | ||
| TA98 | TA100 | TA1535 | TA1537 | E. coli | |||
| Average | 27.1 | 114.7 | 12 | 8.8 | 34.2 | ||
| +S9 | SD | 4 | 19.3 | 1.4 | 2.1 | 5.2 | |
| Minimum | 15 | 71 | 4 | 3 | 16 | ||
| Maximum | 48 | 161 | 24 | 20 | 56 | ||
| Bacterial strains | |||||||
| Historical control data of Water control | TA98 | TA100 | TA1535 | TA1537 | E. coli | ||
| Average | 22.4 | 105.5 | 10.4 | 7.5 | 26.3 | ||
| -S9 | SD | 3.6 | 27.6 | 1.6 | 2.3 | 5.9 | |
| Minimum | 12 | 67 | 3 | 2 | 13 | ||
| Maximum | 36 | 156 | 24 | 15 | 47 | ||
| TA98 | TA100 | TA1535 | TA1537 | E. coli | |||
| Average | 28 | 117.4 | 11.5 | 8.7 | 35.2 | ||
| +S9 | SD | 4 | 19.8 | 1.4 | 2.3 | 5.2 | |
| Minimum | 15 | 83 | 4 | 4 | 18 | ||
| Maximum | 43 | 166 | 22 | 16 | 56 | ||
| Bacterial strains | |||||||
| Historical control data of positive controls | TA98 | TA100 | TA1535 | TA1537 | E. coli | ||
| Average | 255.6 | 958.9 | 842.1 | 467.4 | 712.3 | ||
| -S9 | SD | 30.7 | 149.9 | 134 | 105.7 | 57.5 | |
| Minimum | 123 | 522 | 354 | 109 | 320 | ||
| Maximum | 647 | 1927 | 1871 | 1498 | 1283 | ||
| TA98 | TA100 | TA1535 | TA1537 | E. coli | |||
| Average | 1224.8 | 1431.9 | 165.4 | 148 | 264.7 | ||
| +S9 | SD | 293.8 | 339.9 | 35.1 | 21.3 | 74.2 | |
| Minimum | 409 | 581 | 85 | 68 | 141 | ||
| Maximum | 2587 | 2923 | 507 | 407 | 487 | ||
Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA
SD: Standard deviation; DMSO: Dimethyl sulfoxide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicity in vitro
Ames
The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.
Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:
5000; 1600; 500; 160; 50 and 16 μg/plate.
In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 9.24. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solution (50 mg/mL) were 9.39 and 9.18. The pH of the test item containing overlays was in the range of 7.44 - 7.50 in both experiments. In this study the alkaline test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Micronucleus
A study according to OECD 487 was performed to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor
The test item was dissolved in minimal culture medium to prepare a stock solution (20 mg/mL), corresponding to the highest concentration (2 mg/mL) in the test. In addition, a geometric series of dilutions was prepared from the stock solution.
Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. Cytotoxicity and level of micronuclei were determined. After the culture harvest time, the cells were harvested and slides were prepared. Then, the proportion of cells containing micronuclei was determined.
Two valid independent experiments (experiment I and experiment II) were performed. In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with medium control, solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected.
No cytotoxic effect was detected at any of the tested concentrations in both experiments.
Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed.
All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.
In conclusion, under the experimental conditions reported, the test item does not induce the formation of micronuclei in human lymphocytes in vitro.Therefore, the test item is considered as "not genotoxic" under the conditions of the test.
HPRT
A study according to OECD 476 was performed to investigate the potential of the test item to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X Chinese Hamster V79 cells.
The assay comprised a pre-test and three independent experiments (experiment I and two experiments II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.
The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The two second experiments (experiment IIa and IIb) were performed with a treatment period of 24 hours without metabolic activation.
The following nominal concentrations of the test item were investigated: 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL and 0.06 mg/mL.
The highest nominal concentration (2 mg/mL) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Precipitation of the test item was not observed up to the highest tested concentration.
EMS (experiment I: 300 µg/mL, both experiments II: 150 µg/mL) and DMBA (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies but without reduction of the viability. Thus, they demonstrated enough sensitivity of the testing procedure and the activity of the metabolic activation system.
No cytotoxic effects were observed in any of the test item concentrations in experiment I. Therefore, all concentrations were analyzable for mutagenicity.
In the approach with metabolic activation (experiment I) no dose dependant effect was observed. The values of the solvent and positive control remained well within the range of the historical data. At the highest test item concentration (2 mg/mL), no significant increase in mutant frequency was detected. However, at the 2 next lower test item concentrations (1 mg/mL and 0.5 mg/mL) a discrepancy between the results of replicate A and B was noticed. At the concentration 1 mg/mL a significant increase in mutant frequency was observed at replicate A. At the concentration 0.5 mg/mL the mutant frequency of replicate B induced a significantly increased value. At the test item concentrations 0.25 mg/mL and 0.13 mg/mL no considerable effect was observed. At the lowest test item concentration a significant effect was detected in replicate A but not in replicate B.
A similar effect was observed in the approach without metabolic activation. Again the values of the solvent and positive control remained well within the range of the historical data but for example at the highest test item concentration (2 mg/mL) replicate B induced a strong increase in mutant colonies whereas no increase was detected in replicate A. At the next lower test item concentration (1 mg/mL) the mutant frequency was significantly increased in both replicates. At the next lower test item concentration (0.5 mg/mL) no significant increase was detected. However, at the test item concentration 0.25 mg/mL and 0.13 mg/mL, again the values fluctuate between low mutant frequencies as well as significantly increased mutant frequencies. At the lowest test item concentration both replicates induced a statistically significant increase in mutant frequency.
In experiment IIa the viability was reduced but still within the non-cytotoxic range. Therefore in this experiment the four highest test item concentrations were evaluated. In the highest test item concentration the mutant frequency was significantly increased in replicate B whereas the value of replicate A remained within the historical data. At the next lower test item concentration no effect was detected. At the two lower test item concentrations (0.5 mg/mL and 0.25 mg/mL) one replicate respectively induced a significant in-crease in mutant frequency but the second replicate did not induce such an effect.
In those experiments (experiment I and IIa) no dose-dependent effect was observed. In addition the differences between the concentrations and replicates indicate that there is no obvious mutagenic effect.
Nevertheless, because of the large differences between the single replicates, especially in experiment IIa, a second experiment II (experiment IIb) was performed, to verify the results of experiment IIa and to determine their biologically relevance.
In experiment IIb no cytotoxic effects were observed in any of the test item concentrations. No statistically significant increase in mutant colony numbers/10^6 cells was observed up to the maximum concentration in replicate A, B as well as in the mean values. All values remained well within the historical control range and a dose-dependent effect was not detected. Therefore, the result of experiment IIb is clearly negative.
In accordance with the OECD 476, in experiment I (-S9) and IIa the criteria for a clearly positive or a clearly negative result are not fulfilled. In experiment I (+S9) as well as in experiment IIb the result is clearly negative.
Since the equivocal results in the approach without metabolic activation in experiment I and IIa could not be confirmed in a third experiment (experiment IIb), they are declared as biologically not relevant. Therefore, the overall result of this HPRT test is considered negative.
In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce a positive result in the HPRT test. Therefore, the result of the HPRT test with the test item is considered to be "non-mutagenic under the conditions of the HPRT assay".
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) No 2022/692.
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