Registration Dossier
Registration Dossier
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EC number: 205-351-5 | CAS number: 139-07-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Based on the QSAR predictions for the test substance together with the results of an in vitro Ames test, mammalian cytogenicity and mammalian gene mutation assays with read across substance, the test substance, C12 ADBAC, can be considered to be non-genotoxic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Derek Nexus v6.0.1 profiling
- Type of information:
- (Q)SAR
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- QSAR prediction from a well-known and acknowledged tool. See below 'attached background material section' for detailed prediction results and applicability domain evaluation.
- Qualifier:
- according to guideline
- Guideline:
- other: REACH guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
- Principles of method if other than guideline:
- Since the test substance is a mono constituent substance, the mutagenicity (Ames test) was predicted for the main constituent C12 ADBAC using Derek Nexus v6.0.1 profiling.
- GLP compliance:
- no
- Type of assay:
- other: QSAR prediction
- Key result
- Species / strain:
- other: Mutagenicity in vitro in bacterium prediction using Derek Nexus v6.0.1 profiling
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Based on the prediction for the main constituent in the in vitro bacterial mutagenicity test using the DEREK NEXUS v.6.0.1 expert-rule QSAR model, the test substance is considered to be non-mutagenic.
- Executive summary:
The mutagenicity potential of the test substance C12 ADBAC was predicted using the expert rule-based in silico tool DEREK NEXUS tool v6.0.1. Since the test substance is a mono-constituent, the mutagenicity potential was predicted for the main constituent, using SMILES as the input parameter. As per the tool: “the query structure (i.e., the main constituent) does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test”. Therefore, based on the prediction for the main constituent in the in vitro bacterial mutagenicity test using the DEREK NEXUS v.6.0.1 expert-rule QSAR model, the test substance is considered to be non-mutagenic (Lhasa Ltd., 2019).
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- VEGA-QSAR version 1.1.4
- Type of information:
- (Q)SAR
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- QSAR prediction from a well-known and acknowledged tool. See below 'attached background material section' for detailed prediction results and applicability domain evaluation.
- Qualifier:
- according to guideline
- Guideline:
- other: REACH guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals
- Principles of method if other than guideline:
- Since the test substance is a mono constituent substance, the mutagenicity (Ames test) was predicted for the main constituent C12 ADBAC.
- GLP compliance:
- no
- Type of assay:
- other: QSAR prediction
- Key result
- Species / strain:
- other: QSAR prediction from Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program
- Remarks:
- Ames Mutagenicity Test
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Remarks:
- the predicted compound could be out of the Applicability Domain of the model:
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Based on the prediction for the main constituent using the Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program, the test substance is overall considered to be non-mutagenic.
- Executive summary:
The mutagenicity potential of the test substance C12 ADBAC was predicted using the CAESAR mutagenicity (Ames test) model v.2.1.13 of the VEGA v1.2.4 program. Since the test substance is a mono-constituent, the mutagenicity potential was predicted for the main constituent, using SMILES as the input parameter. The constituent was predicted to be non-mutagenic (VEGA, 2019). The applicability domain of the predictions is typically assessed using an Applicability Domain Index (ADI) that has values from 0 (worst case) to 1 (best case). A global ADI is calculated by grouping several other indices such as similar molecules with known experimental value, accuracy of prediction for similar molecules, concordance for similar molecules, model's descriptors range check and atom centered fragments similarity checks. Usually values lower than 0.75 indicate that the similar compound has important differences compared to the target. For the test substance, the similarity index, accuracy index, concordance index, descriptors range check and ACF index were calculated to be 0.812, 1, 0.696, ‘true’ and 1 respectively leading to global ADI of 0.823. Considering the calculated global ADI for the main constituent, the mutagenicity prediction for the test substance is considered to be reliable with moderate confidence.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From February 28, 2001 to March 26, 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Physical state: Clear liquid
- Analytical purity: The purity of the a.s. is typically >93% ADBAC as described in Section 2; the a.s. is supplied in aqueous/alcohol solution of 50% or 80% a.s. Doses are based on a.s. (i.e. corrected for the dilution in alcohol/water)
- Impurities (identity and concentrations): 0.8% Free Amine + Amine hydrochloride and ≤ 0.1% AAS
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in the dark
- pH (1% water): 6.5 - Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone/β-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (With and without metabolic activation)
Main Experiment: Experiment 1 & 2: 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate (With and without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile distilled water
- Untreated negative controls:
- yes
- Remarks:
- (concurrent untreated)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: Nitroquinoline-1-oxide (4NQO)
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA)
- Remarks:
- With metabolic activation
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 1,8-Dihydroxyanthraquinone (DANTHRON)
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- Method of application: In agar (direct plate incorporation)
Number of replications: Triplicates - Evaluation criteria:
- The test substance was considered positive in the test system if the following criteria were met:
The test substance should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria. - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- - The test substance caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 15µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between experiment number, strain type and exposures with or without S9-mix. The test substance was, therefore, tested up to the toxic limit. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Conclusions:
- Under the study conditions, the test substance was found to be non-mutagenic in Ames test with and without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the test substance, C12 -16 ADBAC (50 -80% active) in an Ames test, according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 850.5100, in compliance with GLP. The mutagenic potential was investigated in Salmonella typhimurium strains A1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance-induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic in Ames test with and without metabolic activation (Thompson, 2001).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From March 22, 2001 to September 25, 2001
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- - Physical state: Extremely pale yellow, slightly viscous liquid with white precipitate
- Analytical purity: The purity of the a.s. is typically >93% ADBAC as described in Section 2; the a.s. is supplied in aqueous/alcohol solution of 50% or 80% a.s. Doses are based on a.s. (i.e. corrected for the dilution in alcohol/water)
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in darkness - Species / strain / cell type:
- lymphocytes: human
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone and ß-naphthoflavone - induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (with and without activation)
Chromosome aberration test:
Experiment 1: 0, 4, 8, 16, 20 µg/mL (with and without activation)
Experiment 2: 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) - Vehicle / solvent:
- Eagle’s minimal essential medium with HEPES buffer (MEM)
- Untreated negative controls:
- yes
- Remarks:
- Negative (media) control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Eagle’s minimal essential medium with HEPES buffer (MEM)
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- (Without S9, at 0.4 and 0.25 µg/mL in Experiment 1 and 2 respectively)
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- (With S9, at 12.5 and 10.0 µg/mL in experiment 1 and 2 respectively)
- Details on test system and experimental conditions:
- Method of application: In Eagle’s minimal essential medium with HEPES buffer (MEM)
Duration:
- Exposure duration:
Without metabolic activation: 4 and 24h
With metabolic activation: 4h
- Exposure procedure: The cultures were incubated at 37˚C for 4 or 24h (as appropriate) in the presence of the test substance at predetermined concentrations/vehicle/positive controls with or without the S9 reaction mixture.
- Expression time: Approximately 20h after initiation of treatment
- Fixation time: 4h
Spindle inhibitor: Demecolcine (colcemid, 0.1 μg/mL) was added approx. 2 h prior to harvest timeSpindle
Stain: When the slides were dry they were stained in 5% Gurrs Giemsa solution for 5 minutes, rinsed, dried and coverslipped using mounting medium.
Number of replications: At least 2 slides/ flask
Number of cell evaluated: 100 consecutive well-spread metaphase cells (if possible), from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.
Determination of the cytotoxicity: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. - Statistics:
- The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test and chi-squared test.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slightly toxic at 20 µg/mL in experiment 1 (with S9 activation) and toxic at 16 µg/mL in experiment 1 and at 20 µg/mL in experiment 2 (without S9 activation))
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Based on the results of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (50 -80% active) in chromosome aberration test, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Based on the results of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001).
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From May 11, 1987 to September 28, 1988
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Alkyldimethylbenzylammonium chloride (ADBAC)
- Physical state: Slightly viscous, clear yellow liquid
- Analytical purity: 81.09% active substance in aqueous/ethanol solution.
- Impurities (identity and concentrations): 0.2% Free amine and 0.1% amine hydrochloride
- Lot/batch No.: 7293K
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions. - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Cell Type: CHO-K1-BH4
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor-induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity assay: 1, 2, 5, 10, 20, 50, 100, 200, 500 and 5000 µg/mL (With and without metabolic activation)
Mutation assay (Without S-9)
Trial I: 1, 5, 10, 13, 16, 20, 25, 35, 50, and 65 µg/mL
Trial II: 1, 5, 10, 12, 14, 16, 18, 20, and 24 µg/mL
Mutation assay (With S-9)
Trial I: 1, 5, 10, 20, 30, 40, 50, 65, 85 and 100 µg/mL
Trial II: 10, 20, 22, 24, 26, 28, 30, 40 and 50 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile deionized water
- Untreated negative controls:
- yes
- Remarks:
- (Negative (media) controls)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (10% water in culture medium)
- Positive controls:
- yes
- Positive control substance:
- other: 5-Bromo-2’-deoxyuridine (BrdU)
- Remarks:
- (without metabolic activation)
- Positive controls:
- yes
- Positive control substance:
- other: 3-Methycholanthrene (MCA)
- Remarks:
- (with metabolic activation)
- Details on test system and experimental conditions:
- Method of applicfation: Single monolayer culture of CHO cells (in medium).
Duration:
- Exposure duration: 4h (with and without metabolic activation)
- Exposure procedure: The cells were quantitatively seeded at 200 cells/dish (cytotoxicity assay) and at about 3 X 10 (6) cells/T-75 (75 cm2) tissue culture flask on the day before dosing. After18h, cell cultures were exposed to test or control substances for 4h.
- Expression time: Approximately 7d after initiation of treatment
- Fixation time: 7d after treatment initiation.
Stain: Colonies were fixed in alcohol, stained with Giemsa solution and counted by eye, excluding those with approximately 50 cells or less.
Number of cells evaluated: 200 cells/dish
Determination of cytotoxicity
- Method: Following parameters were calculated in the study:
1) Relative survival to treatment: The average number of colonies in three dishes (seeded at 200 cells each) was determined.
Relative survival (%) = (Average number of colonies per treated culture/average number of colonies per vehicle control dish) X 100%
2) Relative population growth: This parameter showed the cumulative growth of the treated cell population, relative to the vehicle control growth, over the entire expression period and prior to mutant selection.
Relative population growth (%) = (Treated culture population increase over the expression period/vehicle control population increase over the expression period) X 100%
3) Absolute cloning efficiency: The ability of the cells to form colonies at the time of mutant selection is measured by the absolute cloning efficiency (CE).
Absolute CE (%) = [Average number of viable colonies per dish/200] x 100%
4) Mutant frequency: The mutant frequency is calculated as the ratio of colonies found in thioguanine-selection medium to the total number of cells seeded, adjusted by the absolute CE.
Mutant Frequency = Total mutant clones/[number of dishes X 2x10(5) X absolute CE] - Evaluation criteria:
- Evaluation of a positive response:
To evaluate the test substance as a mutagen following criterion should be met:
- The mutant frequency must meet or exceed 15 x 10E-6 in order to compensate for random fluctuations in the 0 to 10E-6 background mutant frequencies that are typical for this assay.
- A dose related or toxicity related increase in mutant frequency for at least 3 doses should be observed.
- If an increase in mutant frequency is observed for a single dose near the highest testable toxicity, as defined previously and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test substance generally will be considered mutagenic.
Evaluation of a Negative Response:
A test substance is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to concentrations causing about 10 to 15% survival or extends to a concentration at least 75% of that causing excessive toxicity. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- completely toxic at 20 µg/mL and higher concentration (without activation) and at 40 µg/mL and higher concentration (with activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- - Mutant frequencies of all cultures treated with the test material were within the acceptable range for background mutant frequencies (0 to 15E-06 without S9 mix and 0 to 13.5E-06 with S9 mix).
- Mutation Frequency/10(6) cells for trial I and II (without S9 mix) were as follows:
Trial I:
Control: 0, 7.2
Treated: 0 – 4.0
Positive Control (BrdU): 133.3
Trial II:
Control: 8.9, 2.9
Treated: 0.8 – 14.6 (all within historical control range)
Positive Control (BrdU): 114.0
- Mutation Frequency/10(6) cells for trial I and II (with S9 mix) were as follows:
Trial I:
Control: 6.9, 0
Treated: 0.8 – 6.6
Positive Control (3-MCA): 235.3
Trial II:
Control: 1.3, 2.9
Treated: 0.8 – 6.5 (all within historical control range)
Positive Control (3-MCA): 131.7 - Conclusions:
- Based on the results of the study, the test substance is not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) in a mammalian cell gene mutation test, according to a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at test substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the test substance to be slightly more toxic without S9 metabolic activation than with activation. The test substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Based on the results of the study, the test substance is not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989).
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- From February 28, 2001 to March 26, 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone/β-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (With and without metabolic activation)
Main Experiment: Experiment 1 & 2: 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate (With and without metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile distilled water
- Untreated negative controls:
- yes
- Remarks:
- (concurrent untreated)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: Nitroquinoline-1-oxide (4NQO)
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA)
- Remarks:
- With metabolic activation
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 1,8-Dihydroxyanthraquinone (DANTHRON)
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- Method of application: In agar (direct plate incorporation)
Number of replications: Triplicates - Evaluation criteria:
- The test substance was considered positive in the test system if the following criteria were met:
The test substance should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria. - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- - The test substance caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 15µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between experiment number, strain type and exposures with or without S9-mix. The test substance was, therefore, tested up to the toxic limit. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Conclusions:
- Based on the results of the read across substance, the test substance, C12 ADBAC is considered to be non-mutagenic with and without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12 -16 ADBAC (50 -80% active) in an Ames test, according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 850.5100, in compliance with GLP. The mutagenic potential was investigated in Salmonella typhimurium strains A1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance-induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Thompson, 2001). Based on the results of the read across substance, the test substance can also be considered to be non-mutagenic in bacteria with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From March 22, 2001 to September 25, 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: human
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone and ß-naphthoflavone - induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (with and without activation)
Chromosome aberration test:
Experiment 1: 0, 4, 8, 16, 20 µg/mL (with and without activation)
Experiment 2: 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) - Vehicle / solvent:
- Eagle’s minimal essential medium with HEPES buffer (MEM)
- Untreated negative controls:
- yes
- Remarks:
- Negative (media) control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Eagle’s minimal essential medium with HEPES buffer (MEM)
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- (Without S9, at 0.4 and 0.25 µg/mL in Experiment 1 and 2 respectively)
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- (With S9, at 12.5 and 10.0 µg/mL in experiment 1 and 2 respectively)
- Details on test system and experimental conditions:
- Method of application: In Eagle’s minimal essential medium with HEPES buffer (MEM)
Duration:
- Exposure duration:
Without metabolic activation: 4 and 24h
With metabolic activation: 4h
- Exposure procedure: The cultures were incubated at 37˚C for 4 or 24h (as appropriate) in the presence of the test substance at predetermined concentrations/vehicle/positive controls with or without the S9 reaction mixture.
- Expression time: Approximately 20h after initiation of treatment
- Fixation time: 4h
Spindle inhibitor: Demecolcine (colcemid, 0.1 μg/mL) was added approx. 2 h prior to harvest timeSpindle
Stain: When the slides were dry they were stained in 5% Gurrs Giemsa solution for 5 minutes, rinsed, dried and coverslipped using mounting medium.
Number of replications: At least 2 slides/ flask
Number of cell evaluated: 100 consecutive well-spread metaphase cells (if possible), from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.
Determination of the cytotoxicity: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. - Statistics:
- The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test and chi-squared test.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slightly toxic at 20 µg/mL in experiment 1 (with S9 activation) and toxic at 16 µg/mL in experiment 1 and at 20 µg/mL in experiment 2 (without S9 activation))
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Based on the results of the read across study, the test substance, C12 ADBAC, can be considered to be non-clastogenic in mammlian cells with and without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12 -16 ADBAC (50 -80% active) in a chromosome aberration test, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Under the conditions of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001). Based on the results of the read across study, the test substance can also be considered to be non-clastogenic in mammlian cells with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From May 11, 1987 to September 28, 1988
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Cell Type: CHO-K1-BH4
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor-induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity assay: 1, 2, 5, 10, 20, 50, 100, 200, 500 and 5000 µg/mL (With and without metabolic activation)
Mutation assay (Without S-9)
Trial I: 1, 5, 10, 13, 16, 20, 25, 35, 50, and 65 µg/mL
Trial II: 1, 5, 10, 12, 14, 16, 18, 20, and 24 µg/mL
Mutation assay (With S-9)
Trial I: 1, 5, 10, 20, 30, 40, 50, 65, 85 and 100 µg/mL
Trial II: 10, 20, 22, 24, 26, 28, 30, 40 and 50 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile deionized water
- Untreated negative controls:
- yes
- Remarks:
- (Negative (media) controls)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (10% water in culture medium)
- Positive controls:
- yes
- Positive control substance:
- other: 5-Bromo-2’-deoxyuridine (BrdU)
- Remarks:
- (without metabolic activation)
- Positive controls:
- yes
- Positive control substance:
- other: 3-Methycholanthrene (MCA)
- Remarks:
- (with metabolic activation)
- Details on test system and experimental conditions:
- Method of applicfation: Single monolayer culture of CHO cells (in medium).
Duration:
- Exposure duration: 4h (with and without metabolic activation)
- Exposure procedure: The cells were quantitatively seeded at 200 cells/dish (cytotoxicity assay) and at about 3 X 10 (6) cells/T-75 (75 cm2) tissue culture flask on the day before dosing. After18h, cell cultures were exposed to test or control substances for 4h.
- Expression time: Approximately 7d after initiation of treatment
- Fixation time: 7d after treatment initiation.
Stain: Colonies were fixed in alcohol, stained with Giemsa solution and counted by eye, excluding those with approximately 50 cells or less.
Number of cells evaluated: 200 cells/dish
Determination of cytotoxicity
- Method: Following parameters were calculated in the study:
1) Relative survival to treatment: The average number of colonies in three dishes (seeded at 200 cells each) was determined.
Relative survival (%) = (Average number of colonies per treated culture/average number of colonies per vehicle control dish) X 100%
2) Relative population growth: This parameter showed the cumulative growth of the treated cell population, relative to the vehicle control growth, over the entire expression period and prior to mutant selection.
Relative population growth (%) = (Treated culture population increase over the expression period/vehicle control population increase over the expression period) X 100%
3) Absolute cloning efficiency: The ability of the cells to form colonies at the time of mutant selection is measured by the absolute cloning efficiency (CE).
Absolute CE (%) = [Average number of viable colonies per dish/200] x 100%
4) Mutant frequency: The mutant frequency is calculated as the ratio of colonies found in thioguanine-selection medium to the total number of cells seeded, adjusted by the absolute CE.
Mutant Frequency = Total mutant clones/[number of dishes X 2x10(5) X absolute CE] - Evaluation criteria:
- Evaluation of a positive response:
To evaluate the test substance as a mutagen following criterion should be met:
- The mutant frequency must meet or exceed 15 x 10E-6 in order to compensate for random fluctuations in the 0 to 10E-6 background mutant frequencies that are typical for this assay.
- A dose related or toxicity related increase in mutant frequency for at least 3 doses should be observed.
- If an increase in mutant frequency is observed for a single dose near the highest testable toxicity, as defined previously and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test substance generally will be considered mutagenic.
Evaluation of a Negative Response:
A test substance is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to concentrations causing about 10 to 15% survival or extends to a concentration at least 75% of that causing excessive toxicity. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- completely toxic at 20 µg/mL and higher concentration (without activation) and at 40 µg/mL and higher concentration (with activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- - Mutant frequencies of all cultures treated with the test material were within the acceptable range for background mutant frequencies (0 to 15E-06 without S9 mix and 0 to 13.5E-06 with S9 mix).
- Mutation Frequency/10(6) cells for trial I and II (without S9 mix) were as follows:
Trial I:
Control: 0, 7.2
Treated: 0 – 4.0
Positive Control (BrdU): 133.3
Trial II:
Control: 8.9, 2.9
Treated: 0.8 – 14.6 (all within historical control range)
Positive Control (BrdU): 114.0
- Mutation Frequency/10(6) cells for trial I and II (with S9 mix) were as follows:
Trial I:
Control: 6.9, 0
Treated: 0.8 – 6.6
Positive Control (3-MCA): 235.3
Trial II:
Control: 1.3, 2.9
Treated: 0.8 – 6.5 (all within historical control range)
Positive Control (3-MCA): 131.7 - Conclusions:
- Based on the results of the read across study, the test substance, C12 ADBAC is not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) in a cell gene mutation test, according to a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at test substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the test substance to be slightly more toxic without S9 metabolic activation than with activation. The test substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Under study conditions, C12 -16 ADBAC was not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989). Based on the results of the read across substance study, the test substance can also be considered not to induce mutation in the mammalian cells with and without metabolic activation.
Referenceopen allclose all
For the main constituent, the DEREK predicted: ‘No misclassified or unclassified features’.
Details: The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test.
Prediction is NON-Mutagenic, but the result shows some critical aspects, which require to be checked:
- only moderately similar compounds with known experimental value in the training set have been found
- some similar molecules found in the training set have experimental values that disagree with the predicted value
Table 1. Cytotoxicity (Number of revertant colonies)
|
|
Test substance concentration (µg/plate) |
||||||||||
With/ Without S9-mix |
Strain |
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
Without |
TA100 |
91 |
91 |
81 |
83 |
64 |
17S |
0T |
0T |
0T |
0T |
0T |
With |
TA100 |
97 |
105 |
103 |
103 |
83 |
50S |
0T |
0T |
0T |
0T |
0T |
S=sparse bacterial background lawn
T= toxic, no bacterial lawn
Table 2. Genotoxicity (Mean number of revertant colonies)
Strain |
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||||||
Test substance concentration (mg/plate) |
|
|
|
|
|
||||||
With S9 |
|
|
|
|
|
||||||
+ve control type (concentration (mg/plate)) |
2AA (1) |
2AA (2) |
DAN (10) |
BP (5) |
2AA (2) |
||||||
Test number |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
|
+ve control |
1772 |
2317 |
287 |
135 |
886 |
723 |
229 |
251 |
582 |
336 |
|
-ve control |
143 |
137 |
17 |
17 |
349 |
308 |
36 |
25 |
12 |
22 |
|
0.15 |
129 |
137 |
13 |
13 |
357 |
315 |
26 |
24 |
13 |
18 |
|
0.5 |
134 |
11 |
10 |
14 |
346 |
313 |
33 |
25 |
15 |
14 |
|
1.5 |
129 |
127 |
15 |
14 |
373 |
307 |
32 |
26 |
16 |
14 |
|
5 |
142 |
143 |
10 |
13 |
369 |
341 |
32 |
23 |
17 |
16 |
|
15 |
132 |
0 |
12 |
2 |
363 |
151 |
37 |
11 |
17 |
6 |
|
50 |
28 |
0 |
10 |
0 |
276 |
0 |
22 |
0 |
10 |
0 |
|
Without S9 |
|
|
|
|
|
||||||
+ve control type (concentration (mg/plate)) |
ENNG (3) |
ENNG (5) |
MMC (0.5) |
4NQO (0.2) |
9AA (80) |
||||||
Test number |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
|
+ve control |
621 |
464 |
603 |
430 |
854 |
961 |
142 |
126 |
656 |
716 |
|
-ve control |
153 |
134 |
12 |
18 |
316 |
336 |
38 |
22 |
16 |
17 |
|
0.15 |
150 |
123 |
13 |
15 |
340 |
308 |
29 |
18 |
18 |
19 |
|
0.5 |
132 |
114 |
11 |
21 |
331 |
339 |
30 |
18 |
17 |
18 |
|
1.5 |
154 |
111 |
19 |
16 |
339 |
232 |
25 |
14 |
15 |
11 |
|
5 |
145 |
111 |
9 |
10 |
326 |
325 |
28 |
19 |
12 |
18 |
|
15 |
84 |
0 |
11 |
0 |
320 |
0 |
23 |
0 |
0 |
0 |
|
50 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
For further details refer to the attachment under 'attached background material'.
Table 1. Results of chromosomal aberration in human lymphocytes (Experiment 1)
Treatment (μg/mL) |
S9 Activation |
Treatment Time |
Mean Mitotic Index |
Cells Scored |
Total Number of Aberrations |
Cells with Numerical Aberrations + Gaps(%) |
Cells with Structural Aberrations - Gaps(%) |
Vehicle |
- |
4 |
4.2 |
200 |
11 7 |
5.0 |
3.5 |
Test substance |
|||||||
4 |
- |
4 |
3.2 |
200 |
6 1 |
3.0 |
0.5 |
8 |
- |
4 |
2.7 |
200 |
2 1 |
1.0 |
0.5 |
16 |
- |
4 |
1.5 |
200 |
7 5 |
3.0 |
2.0 |
Positive control (MMC) 0.4 |
- |
4 |
2.2 |
200 |
83 64 |
32.0 |
26.0** |
|
|||||||
Vehicle |
+ |
4 |
3.9 |
200 |
5 1 |
2.5 |
0.5 |
Test substance |
|||||||
8 |
+ |
4 |
2.3 |
200 |
4 1 |
2.0 |
0.5 |
16 |
+ |
4 |
2.1 |
200 |
7 4 |
2.5 |
1.0 |
20 |
+ |
4 |
1.6 |
200 |
7 3 |
3.5 |
1.5 |
Positive control (CP) 12.5 |
+ |
4 |
1.4 |
300 |
76 45 |
20.0 |
12.0** |
Treatment: Cells from the 4-h treatment regimens were harvested 20h after the initiation of the treatments.
Frequency of Aberrant Cells: **, p ≤ 0.001
Table 2. Results of chromosomal aberration in human lymphocytes (Experiment 2)
Treatment (μg/mL) |
S9 Activation |
Treatment Time |
Mean Mitotic Index |
Cells Scored |
Total Number of Aberrations |
Cells with Numerical Aberrations + Gaps(%) |
Cells with Structural Aberrations - Gaps(%) |
Vehicle |
- |
4 |
7.0 |
200 |
4 1 |
2.0 |
0.5 |
Test substance |
|||||||
4 |
- |
24 |
4.9 |
200 |
6 4 |
2.0 |
1.0 |
8 |
- |
24 |
2.7 |
200 |
6 6 |
2.5 |
2.5 |
12 |
- |
24 |
2.6 |
200 |
12 2 |
6.0 |
1.0 |
Positive control (MMC) 0.2 |
- |
24 |
2.3 |
200 |
115 86 |
37.0 |
30.0** |
|
|||||||
Vehicle |
+ |
4 |
5.9 |
200 |
8 4 |
2.5 |
0.5 |
Test substance |
|||||||
8 |
+ |
4 |
5.2 |
200 |
7 3 |
3.5 |
1.5 |
16 |
+ |
4 |
3.3 |
200 |
0 0 |
0 |
0 |
24 |
+ |
4 |
3.4 |
200 |
1 1 |
0.5 |
0.5 |
Positive control (CP) 12.5 |
+ |
4 |
1.4 |
200 |
108 79 |
33.5 |
27.0** |
Treatment: Cells from both the 4-h and 24 h treatment regimens were harvested 20 h after the initiation of the treatments.
Frequency of Aberrant Cells: **, p ≤ 0.001
For further details, refer to the attachment under 'Attached background material'
For details on results, please refer to the attachment under 'Attached background material'
Table 1. Cytotoxicity (Number of revertant colonies)
|
|
Test substance concentration (µg/plate) |
||||||||||
With/ Without S9-mix |
Strain |
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
Without |
TA100 |
91 |
91 |
81 |
83 |
64 |
17S |
0T |
0T |
0T |
0T |
0T |
With |
TA100 |
97 |
105 |
103 |
103 |
83 |
50S |
0T |
0T |
0T |
0T |
0T |
S=sparse bacterial background lawn
T= toxic, no bacterial lawn
Table 2. Genotoxicity (Mean number of revertant colonies)
Strain |
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||||||
Test substance concentration (mg/plate) |
|
|
|
|
|
||||||
With S9 |
|
|
|
|
|
||||||
+ve control type (concentration (mg/plate)) |
2AA (1) |
2AA (2) |
DAN (10) |
BP (5) |
2AA (2) |
||||||
Test number |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
|
+ve control |
1772 |
2317 |
287 |
135 |
886 |
723 |
229 |
251 |
582 |
336 |
|
-ve control |
143 |
137 |
17 |
17 |
349 |
308 |
36 |
25 |
12 |
22 |
|
0.15 |
129 |
137 |
13 |
13 |
357 |
315 |
26 |
24 |
13 |
18 |
|
0.5 |
134 |
11 |
10 |
14 |
346 |
313 |
33 |
25 |
15 |
14 |
|
1.5 |
129 |
127 |
15 |
14 |
373 |
307 |
32 |
26 |
16 |
14 |
|
5 |
142 |
143 |
10 |
13 |
369 |
341 |
32 |
23 |
17 |
16 |
|
15 |
132 |
0 |
12 |
2 |
363 |
151 |
37 |
11 |
17 |
6 |
|
50 |
28 |
0 |
10 |
0 |
276 |
0 |
22 |
0 |
10 |
0 |
|
Without S9 |
|
|
|
|
|
||||||
+ve control type (concentration (mg/plate)) |
ENNG (3) |
ENNG (5) |
MMC (0.5) |
4NQO (0.2) |
9AA (80) |
||||||
Test number |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
1 |
2 |
|
+ve control |
621 |
464 |
603 |
430 |
854 |
961 |
142 |
126 |
656 |
716 |
|
-ve control |
153 |
134 |
12 |
18 |
316 |
336 |
38 |
22 |
16 |
17 |
|
0.15 |
150 |
123 |
13 |
15 |
340 |
308 |
29 |
18 |
18 |
19 |
|
0.5 |
132 |
114 |
11 |
21 |
331 |
339 |
30 |
18 |
17 |
18 |
|
1.5 |
154 |
111 |
19 |
16 |
339 |
232 |
25 |
14 |
15 |
11 |
|
5 |
145 |
111 |
9 |
10 |
326 |
325 |
28 |
19 |
12 |
18 |
|
15 |
84 |
0 |
11 |
0 |
320 |
0 |
23 |
0 |
0 |
0 |
|
50 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Table 1. Results of chromosomal aberration in human lymphocytes (Experiment 1)
Treatment (μg/mL) |
S9 Activation |
Treatment Time |
Mean Mitotic Index |
Cells Scored |
Total Number of Aberrations |
Cells with Numerical Aberrations + Gaps(%) |
Cells with Structural Aberrations - Gaps(%) |
Vehicle |
- |
4 |
4.2 |
200 |
11 7 |
5.0 |
3.5 |
Test substance |
|||||||
4 |
- |
4 |
3.2 |
200 |
6 1 |
3.0 |
0.5 |
8 |
- |
4 |
2.7 |
200 |
2 1 |
1.0 |
0.5 |
16 |
- |
4 |
1.5 |
200 |
7 5 |
3.0 |
2.0 |
Positive control (MMC) 0.4 |
- |
4 |
2.2 |
200 |
83 64 |
32.0 |
26.0** |
|
|||||||
Vehicle |
+ |
4 |
3.9 |
200 |
5 1 |
2.5 |
0.5 |
Test substance |
|||||||
8 |
+ |
4 |
2.3 |
200 |
4 1 |
2.0 |
0.5 |
16 |
+ |
4 |
2.1 |
200 |
7 4 |
2.5 |
1.0 |
20 |
+ |
4 |
1.6 |
200 |
7 3 |
3.5 |
1.5 |
Positive control (CP) 12.5 |
+ |
4 |
1.4 |
300 |
76 45 |
20.0 |
12.0** |
Treatment: Cells from the 4-h treatment regimens were harvested 20h after the initiation of the treatments.
Frequency of Aberrant Cells: **, p ≤ 0.001
Table 2. Results of chromosomal aberration in human lymphocytes (Experiment 2)
Treatment (μg/mL) |
S9 Activation |
Treatment Time |
Mean Mitotic Index |
Cells Scored |
Total Number of Aberrations |
Cells with Numerical Aberrations + Gaps(%) |
Cells with Structural Aberrations - Gaps(%) |
Vehicle |
- |
4 |
7.0 |
200 |
4 1 |
2.0 |
0.5 |
Test substance |
|||||||
4 |
- |
24 |
4.9 |
200 |
6 4 |
2.0 |
1.0 |
8 |
- |
24 |
2.7 |
200 |
6 6 |
2.5 |
2.5 |
12 |
- |
24 |
2.6 |
200 |
12 2 |
6.0 |
1.0 |
Positive control (MMC) 0.2 |
- |
24 |
2.3 |
200 |
115 86 |
37.0 |
30.0** |
|
|||||||
Vehicle |
+ |
4 |
5.9 |
200 |
8 4 |
2.5 |
0.5 |
Test substance |
|||||||
8 |
+ |
4 |
5.2 |
200 |
7 3 |
3.5 |
1.5 |
16 |
+ |
4 |
3.3 |
200 |
0 0 |
0 |
0 |
24 |
+ |
4 |
3.4 |
200 |
1 1 |
0.5 |
0.5 |
Positive control (CP) 12.5 |
+ |
4 |
1.4 |
200 |
108 79 |
33.5 |
27.0** |
Treatment: Cells from both the 4-h and 24 h treatment regimens were harvested 20 h after the initiation of the treatments.
Frequency of Aberrant Cells: **, p ≤ 0.001
For further details, refer to the attached document under 'Attached background material'
For details on results, please refer to the attachment under 'Attached background material'
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro Bacterial mutagenicity test:
Study 1: The mutagenicity potential of the test substance C12 ADBAC was predicted using the expert rule-based in silico tool DEREK NEXUS tool v6.0.1. Since the test substance is a mono-constituent, the mutagenicity potential was predicted for the main constituent, using SMILES as the input parameter. As per the tool: “the query structure (i.e., the main constituent) does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be inactive in the bacterial in vitro (Ames) mutagenicity test”. Therefore, based on the prediction for the main constituent in the in vitro bacterial mutagenicity test using the DEREK NEXUS v.6.0.1 expert-rule QSAR model, the test substance is considered to be non-mutagenic (Lhasa Ltd., 2019). The expert-rule prediction using DEREK NEXUS is considered to be reliable with high confidence.
Study 2: The mutagenicity potential of the test substance C12 ADBAC was predicted using the Mutagenicity (Ames test) model (CAESAR) 2.1.13 of the VEGA v1.2.4 program. Since the test substance is a mono-constituent, the mutagenicity potential was predicted for the main constituent, using SMILES as the input parameter. The constituent was predicted to be non-mutagenic (VEGA, 2019). The applicability domain of the predictions is typically assessed using an Applicability Domain Index (ADI) that has values from 0 (worst case) to 1 (best case). A global ADI is calculated by grouping several other indices such as similar molecules with known experimental value, accuracy of prediction for similar molecules, concordance for similar molecules, model's descriptors range check and atom centered fragments similarity checks. Usually values lower than 0.75 indicate that the similar compound has important differences compared to the target. For the test substance, the similarity index, accuracy index, concordance index, descriptors range check and ACF index were calculated to be 0.812, 1, 0.696, ‘true’ and 1 respectively leading to global ADI of 0.823. Considering the calculated global ADI for the main constituent, the mutagenicity prediction for the test substance is considered to be reliable with moderate confidence.
Study 3: A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12 -16 ADBAC (50 -80% active), according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 850.5100 (Ames test), in compliance with GLP. The mutagenic potential was investigated in Salmonella typhimurium strains A1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance-induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Thompson, 2001). Based on the results of the read across substance, the test substance can also be considered to be non-mutagenic in bacteria with and without metabolic activation.
Based on the QSAR prediction as well as the experimental results from the in vitro study with the read across substance, the test substance is overall considered to be non-mutagenic in bacteria.
In vitro mammalian mutagenicity test:
Study 1: A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution)in amammalian cell gene mutation assay, according to a method similar to US EPA OPPTS 870.5300, in compliance with GLP. The study was performed on the HGPRT locus in Chinese hamster ovary (CHO) cells at test substance concentrations ranging from 0 to100 µg/mL. Preliminary cytotoxicity test showed the test substance to be slightly more toxic without S9 metabolic activation than with activation. The test substance was completely toxic at 20 µg/mL and higher without activation and completely toxic at 40 µg/mL and higher with activation. Dose levels selected for the first trial of the mutation assays covered nontoxic and highly toxic doses. Two independent non-activation and S9 metabolic activation assays were performed. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies (0 to 13.5 x 10-6 with S9 mix and 0 to 15 x 10-6 without S9 mix). Under study conditions, C12 -16 ADBAC was not considered to induce any forward mutations at the HGPRT locus in CHO cells with and without metabolic activation (Young, 1989). Based on the results of the read across substance study, the test substance C12 ADBAC, is considered not to induce mutation in the mammalian cells with and without metabolic activation.
In vitro mammalian cytogenicity test:
Study 1: A study was conducted to determine the in vitro genetic toxicity of the read across substance, C12 -16 ADBAC(50 -80% active),according to OECD Guideline 473 and EU Method B.10 (chromosome aberration test), in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Under the conditions of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001). Based on the results of the read across study, the test substance can also be considered to be non-clastogenic in mammalian cells with and without metabolic activation.
Justification for classification or non-classification
Based on the QSAR predictions for the test substance together with the results of an in vitro Ames test, mammalian cytogenicity and mammalian gene mutation assays with read across substance, it can be concluded that the test substance, C12 ADBAC does not warrant a classification for genotoxicity according to EU CLP criteria (Regulation EC 1272/2008).
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