Registration Dossier
Registration Dossier
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EC number: 939-253-5 | CAS number: 68424-85-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test substance was not mutagenic in bacterial reverse mutation assays or in the HPRT locus in Chinese hamster ovary cells, either in the presence or absence of metabolic activation. The number of chromosomal aberrations was not increased by the test substance in the Chinese hamster ovary cells or human lymphocytes. Further, no increase in the frequency of micronucleated polychromatic erythrocytes was observed in peripheral blood samples from male and female mice treated orally with 400 mg of test substance / kg bw. Based on the overall weight of evidence, the test substance is non-genotoxic.
Link to relevant study records
- 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
- 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
- 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
- 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
- Positive controls validity:
- valid
- 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
- 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
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium 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. - Remarks on result:
- other: not mutagenic
- 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 (49.7% active in water) 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 gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1986
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- other: Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver microsome fraction of Aroclor-induced rats for metabolic activation (S9-mix)
- Test concentrations with justification for top dose:
- 0, 0.31, 0.93, 2.78, 8.33 and 25 µg/plate.
- Details on test system and experimental conditions:
- Salmonella typhimurium reverse mutation assay
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- >= 50.0 µg per plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- >= 50.0 µg per plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- >= 50.0 µg per plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- >= 50.0 µg per plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- >= 50.0 µg per plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: not mutagenic
- Conclusions:
- Under the study conditions, the test substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 471 (Ames test), in compliance with GLP. The substance was examined for mutagenic activity in the Ames test using the histidine-requiring Salmonella typhimurium mutant strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S9-mix). The substance was tested at doses of 0, 0.31, 0.93, 2.78, 8.33 and 25 µg/plate. Based on preliminary test in TA 98, 25 µg/plate was chosen as the highest dose level. The test was carried out twice. Under the study conditions, the substance was considered to be non-mutagenic in the presence and absence of exogenous metabolic activation (Wilmer, 1986).
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- 5 strains were used, but not one of the strains having AT base pairs at the primary reversion site (such as TA 102 or E. coli WP2 strains).
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- reverse mutation to histidine independency
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver (S9-mix)
- Test concentrations with justification for top dose:
- RF test: 0, 5, 50, 500, 5000 µg/plate
main test 1: 1.5, 5, 15, 50, 150 µg/plate
main test 2: 0.5, 1.5, 5, 15, 50 µg/plate - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N'-nitro-N-nitroso-guanidine (TA1535 and TA100), 9-aminoacridine (TA1537), 2-nitrofluorene (TA1538 and TA98)
- Remarks:
- without S9-mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (all strains)
- Remarks:
- with S9-mix
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 50 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- not up to 150 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No substantial increases in revertant colony numbers of any of the five tester strains were observed; the test substance was found to be non-mutagenic in the Ames test using strains TA1535, TA1537, TA1538, TA98 and TA100. Although these strains may not detect certain oxidising mutagens or cross-linking agents, it is not expected that the test substance will do so. Therefore, it is concluded that there is no evidence of mutagenic potential of the test substance in this bacterial test system.
- Remarks on result:
- other: not mutagenic
- Conclusions:
- Under the study conditions, no significant increase in the numbers of revertant colonies of Salmonella typhimurium strains was recorded at any dose, either with or without metabolic activation. Therefore, the test substance was found to be non-mutagenic under the conditions of this test.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 471 (Ames test), in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance by the Ames plate incorporation method at five dose levels, in triplicate, with and without the addition of a rat liver homogenate metabolising system. The dose range was determined in a preliminary toxicity assay using 5 to 5,000 µg/plate. Doses in the first experiment were 1.5 -150 µg/plate. The experiment was repeated on a separate day. In this case, the dose range was 0.5 to 50 µg/plate. The solvent (DMSO) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test substance caused visible reduction in the growth of the bacterial lawn at 50 µg/plate (TA1535, TA100) and at 150 µg/plate (TA1535, TA98 and TA100). The test substance was, therefore, tested up to the maximally 150 µg/plate and 50 µg/plate in the first and second test, respectively. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Jones, 1985).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1986
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from rats treated with Aroclor 1254 (S-9 mix)
- Test concentrations with justification for top dose:
- Based on preliminary cytotoxicity test: 0, 0.36, 1.09, 3.27 and 9.80 µg/mL (without S9); 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254).
- Details on test system and experimental conditions:
- Exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: >= 15.68 µg/mL (- S9); >= 78.4 µg/mL (+S9)
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- - A preliminary toxicity test, showed that in the absence of S9-mix, the substance was toxic in concentrations of 15.68 µg/mL and above; in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times.
- The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. - Remarks on result:
- other: not clastogenic
- Conclusions:
- Under the study conditions, the test substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation.
- Executive summary:
An in vitro study was conducted to determine the clastogenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. This experiment was realized in Chinese hamster ovary cells. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL test substance (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) test substance were selected. The exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the study conditions, the test substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation (Willmer, 1986).
- 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
- 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
- Remarks on result:
- other: not clastogenic
- 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:
An in vitro study was conducted to determine the clastogenic potential of the test substance, C12-16 ADBAC (49.7% active in water) 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:
- 1986
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HGPRT locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Based on a preliminary cytotoxicity test:
0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and
0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) - Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- - The point mutation test was carried out in duplicate both in the presence and the absence of a metabolic activation system.
- Exposure duration of 5h. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: > 17.5 nL/mL (- S9), >= 70 nL/mL (+ S9)
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- - Concentration-dependent increases in the number of mutants at the HGPRT locus were not observed either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to either EMS or DMN gave the expected increases in the mutation frequency.
The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. - Remarks on result:
- other: not mutagenic
- Conclusions:
- Under the study conditions, the test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with or without metabolic activation.
- Executive summary:
An in vitro study was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. This study was performed to evaluate the potential to induce mutations at the HGPRT locus in Chinese hamster ovary (CHO) cells. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five h exposure was used both with and without S9-mix. The test substance did not induce dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was clearly reduced in the presence of metabolic activation. Under the study conditions, test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986).
- 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:
- according to guideline
- Guideline:
- other: U.S. EPA FIFRA 84-4
- Version / remarks:
- 1989
- Deviations:
- no
- 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 - Remarks on result:
- other: not mutagenic
- 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:
An in vitro study was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) in a mammalian cell gene mutation test, according to US EPA FIFRA 84-4 or 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:
- genetic toxicity in vitro
- Type of information:
- other: Published data
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Documentation insufficient for assessment.
- Principles of method if other than guideline:
- Guideline not mentioned. Collated data of various published studies.
- Type of assay:
- other: Collated data of various in vitro studies
- Conclusions:
- Similar studies reported in literature do not give evidence of genotoxicity. However, for test substance also positive, negative and equivocal results were seen in the B. subtilis rec assays. In an E. coli DNA polymerase assay test substance induced repairable DNA damage, which points towards a genetic damage (BIBRA 1989).
- Executive summary:
Similar studies reported in literature do not give evidence of genotoxicity. However, for test substance also positive, negative and equivocal results were seen in the B. subtilis rec assays. In an E. coli DNA polymerase assay test substance induced repairable DNA damage, which points towards a genetic damage (BIBRA 1989).
Referenceopen allclose all
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'.
For result tables, kindly refer to the attached background material section of the IUCLID.
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'
Refer overall remarks and attachments section.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1985
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EEC Directive 79/831, Annex V, Method No. 431
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Test animals
- Source: Breeders were purchased from G1. Bomholtgard Ltd., but the mice used were born in the Scantox Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: Yes, in groups of 5
- Fasting period before study: No
- Housing: 5 animals/cage, males and females separately in type III Macrolone cages, bedding used was special softwood sawdust "Spanvall Special White " from Spanvall Ltd., DK-4535 Vallekilde
- Diet: Complete rodent diet "Altromin 1314" from Chr. Petersen Ltd., DK-4100 Ringsted, ad libitum
- Water: Drinking water adjusted to pH 2.5 with hydrochloric acid, ad libitum
- Acclimation period:
Environmental conditions
- Temperature: 21 ±2°C
- Humidity: 55 ± 15%
- Air changes: 10/h
- Photoperiod: 12h dark /12h light - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Distilled water
- Concentration of test material in vehicle: 400 mg/10 mL distilled water for the dose level of 400 mg/kg bw.
- Amount of vehicle: The vehicle was administered orally at a volume of 10 mL/kg bw. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Dilution of the test substance in distilled water
- Frequency of treatment:
- Once
- Post exposure period:
- 24, 48 and 72 h
- Dose / conc.:
- 400 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide (positive control):
- Route of administration: Oral
- Doses / concentrations: 30 mg/10 mL equivalent to 30 mg/kg bw
- Tissues and cell types examined:
- Bone marrow erythrocytes
- Details of tissue and slide preparation:
- Dose selection:
Preliminary investigations:
- A few mice were treated orally with various concentrations of the test substance diluted with distilled water. Thereby the maximum tolerated dose was estimated at 400 mg/kg bw. At this dosage bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).
Details of slide preparations: Immediately after sacrifice, femurs of a mouse were dissected free of muscle, and by a 1 mL syringe with needle the bone marrow was flushed out into 5 mL of fetal calf serum. After thorough shaking, the mixture was centrifuged for 10 min. at about 1000 rpm. Thereafter, smears were made after removal of the supernatant. The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.
Method of analysis: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind.
The following counts were made:
Number of normochromatic erythrocytes (NCE) per 1000 erythrocytes
Number of polychromatic erythrocytes (PCE) per 1000 erythrocytes
Number of micronuclei (MN) in 1000 normochromatic erythrocytes
Number of micronuclei (MN) in 1000 polychromatic erythrocytes. - Evaluation criteria:
- Increase in the frequency of micronucleated polychromatic erythrocytes in treated animals as compared to controls
- Statistics:
- The statistical difference was analysed by one-way ANOVA. In the case of PCE (%) the test was performed on the values observed, and for the MN (per thousand) the test was done on computed rank values transformed to normal scores according to Blom's method (Blom, 1958).
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- one mortality in the 72h test group on Day 3 after treatment
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: not genotoxic
- Additional information on results:
- Results of the dose-range finding study
- Dose range: Up to 400 mg/kg bw
- Clinical signs of toxicity in test animals: Maximum tolerated dose was estimated at 400 mg/kg bw. At a dose exceeding 400 mg/kg bw, the mortality was too high.
- Evidence of cytotoxicity in tissue analyzed: At 400 mg/kg bw, bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).
Results of the main test
- Induction of micronuclei: No significant difference as compared to controls.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes - Conclusions:
- Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice.
- Executive summary:
A study was conducted to determine the in vivo toxicity of the test substance, C12-16 ADBAC (80.2% active in ethanol) according to OECD Guideline 474, in compliance with GLP. This study was performed to evaluate the chromosome-damaging effect of the test substance in mice. The experimental animals were 50 NMRI mice, divided into 5 groups. Of the 5 groups, three were test groups, one negative control group and one positive control group. The test groups were treated with 400 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen, 1985).
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro
Ames test:
Study 1:Anin vitrostudy was conducted to determine the mutagenic potential of the test substance, C12 -16 ADBAC (49.7% active in water) 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 inSalmonella typhimuriumstrains TA1535, 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 was 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 the Ames test with and without metabolic activation (Thompson, 2001).
Study 2:Anin vitrostudy was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 471 (Ames test), in compliance with GLP. The substance was examined for mutagenic activity in the Ames test using the histidine-requiringSalmonella typhimuriummutant strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S9-mix). The substance was tested at doses of 0, 0.31, 0.93, 2.78, 8.33 and 25 µg/plate. Based on the preliminary test in TA 98, 25 µg/plate was chosen as the highest dose level. The test was carried out twice. Under the study conditions, the substance was considered to be non-mutagenic in the presence and absence of exogenous metabolic activation (Wilmer, 1986).
Study 3:Anin vitrostudy was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 471 (Ames test), in compliance with GLP.Salmonella typhimuriumstrains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance by the Ames plate incorporation method at five dose levels, in triplicate, with and without the addition of a rat liver homogenate metabolising system. The dose range was determined in a preliminary toxicity assay using 5 to 5,000 µg/plate. Doses in the first experiment were 1.5 -150 µg/plate. The experiment was repeated on a separate day. In this case, the dose range was 0.5 to 50 µg/plate. The solvent (DMSO) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test substance caused visible reduction in the growth of the bacterial lawn at 50 µg/plate (TA1535, TA100) and at 150 µg/plate (TA1535, TA98 and TA100). The test substance was, therefore, tested up to the maximally 150 µg/plate and 50 µg/plate in the first and second test, respectively. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation (Jones, 1985).
Chromosomal aberration test
Study 1:An in vitro study was conducted to determine the clastogenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 473 (chromosome aberration test), in compliance with GLP. This experiment was realized in Chinese hamster ovary cells. Based on preliminary cytotoxicity test, 0, 0.36, 1.09, 3.27 and 9.80 µg/mL test substance (without S9) as well as 0, 6.13, 12.25, 24.5, 49.0 and 98.0 µg/mL (with metabolic activation of S9 from rats treated with Aroclor 1254) test substance were selected. The exposure times were 21h for the experiments without metabolic activation, and 3h for the experiments with metabolic activation, with harvesting times of 12 and 21h. In the preliminary toxicity test, in the absence of S9-mix, the substance was toxic at a concentration of 15.68 µg/mL and above; and in the presence of S9-mix, the substance was very toxic at 78.4 µg/mL and above at both harvesting times. The test substance did not induce a statistically significant increase in the number of cells with structural chromosome aberrations (breaks, exchanges and multiple aberrations) at any of the concentrations used, either in the absence or in the presence of the S-9 mix. Under the study conditions, the test substance was not clastogenic in Chinese hamster ovary cells with or without metabolic activation (Willmer, 1986).
Study 2:An in vitro study was conducted to determine the clastogenic potential of the test substance, C12-16 ADBAC (49.7% active in water) 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 the 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).
Mammalian cell gene mutation test:
Study 1:An in vitro study was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (50% active in water) according to OECD Guideline 476 (cell gene mutation test), in compliance with GLP. This study was performed to evaluate the potential to induce mutations at the HGPRT locus in Chinese hamster ovary (CHO) cells. Based on a preliminary toxicity test, 0, 5, 10, 12.5, 15, 17.5 and 20 nL/mL (without metabolic activation) and 0, 15, 30, 45, 60, 75 and 90 nL/mL (with metabolic activation) were selected for the experiment. The entire experiment was repeated to confirm the results of the first experiment. Five h exposure was used both with and without S9-mix. The test substance did not induce a dose-related increase in the mutant frequency at the HGPRT locus at any dose level, either with or without metabolic activation. Exposure to the highest concentrations tested resulted in significant (initial) toxicity for the CHO cells; this indicates that the test was carried out with appropriate concentrations of the test substance. Exposure to positive control substances gave the expected increases in the mutation frequency. The toxicity of the test substance for CHO cells was reduced in the presence of metabolic activation. Under the study conditions, the test substance did not show any mutagenic activity at the HGPRT locus in CHO cells with and without metabolic activation (Davis, 1986).
Study 2:An in vitro study was conducted to determine the mutagenic potential of the test substance, C12-16 ADBAC (81.09% active in aqueous/ethanol solution) in a mammalian cell gene mutation test, according to US EPA FIFRA 84-4 or 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).
Similar studies reported in the literature do not give evidence of genotoxicity. However, for test substance also positive, negative and equivocal results were seen in the B. subtilis rec assays. In an E. coli DNA polymerase assay test substance-induced repairable DNA damage, which points towards genetic damage (BIBRA 1989).
In vivo
Micronucleus assay
An in vivo micronucleus assay was conducted to determine the clastogenic potential of the test substance in mice according to OECD Guideline 474, in compliance with GLP. The experimental animals were 50 NMRI mice, divided into 5 groups. Of the 5 groups, three were test groups, one negative control group and one positive control group. The test groups were treated with 400 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen, 1985).
The biocides assessment reports available from RMS Italy on C12-16 ADBAC, also concluded that the test substance can be considered not genotoxic based onin vitro(Ames test, Chromosomal aberration test, Mammalian cell gene mutation assay) andin vivotest (Chromosomal aberration test in rat bone marrow) (ECHA biocides assessment report, 2015).
Therefore, based on the available information and in line with the biocides assessment report, the test substance is considered have no genotoxic potential.
Justification for classification or non-classification
Based on the results from in vitro and in vivo genotoxicity assays, no classification for genotoxicity is warranted for the test substance according to EU CLP criteria (Regulation EC 1272/2008).
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