Registration Dossier
Registration Dossier
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EC number: 236-740-8 | CAS number: 13472-08-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
All in vitro studies on 13472-08-7 (two Ames), and on read across substance 78 -67 -1 (Ames, MLA, Chrome ab) are negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. GLP Guideline study (OECD 471)
- Justification for type of information:
- see 13.2 for attached read across rationale
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 97
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with phenobarbital and 5,6-benzoflavone.
- Test concentrations with justification for top dose:
- 0, 313, 625, 1250, 2500 and 5000 µg/plate
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: (see below: details on test system and conditions)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- The test was performed according to the pre-incubation method.
NUMBER AND SELECTION OF DOSES, CONTROLS USED
- Five doses of test substance, together with the appropriate concurrent solvent and positive controls, were tested in triplicate on each tester strain with and without metabolic activation. The experiment was repeated twice except for TA1537 whic was tested four times.
-The dose-levels were the following: 0, 313,625, 1250, 2500 and 5000 µg/plate. All the strains were tested without S9 whereas TA97 was not part
of the experiment performed with S9.
The positive controls were as follows:
without S9 mix:
• sodium azide (NaN3): TA 1535 strain
• 9-Aminoacridine (9AA): TA 1537 and TA97 strains,
• 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide: TA 98 and TA 100 strains,
with S9 mix:
• 2-Aminoanthracene: TA 1535, TA 1537, TA 98 and TA 100 strains, - Evaluation criteria:
- Not precised
- Statistics:
- no
- Species / strain:
- S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 97
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- See tables below.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Under the experimental conditions described, the test substance 2,2'-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella or E. coli. - Executive summary:
The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to induce reverse mutation in Salmonella typhimurium was evaluated according to OECD 471 guideline in compliance with the Principles of Good Laboratory Practice.
Methods:
The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with phenobarbital and 5,6-benzoflavone. Both experiments were performed according to the preincubation method. Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 97 were used except for TA 97 which was only used in the experiment without S9. E. coli WP2 uvrA was also tested with and without activation. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item 2,2-AZOBIS(ISOBUTYRONITRILE) was dissolved in dimethylsulfoxide (DMSO) and the following positive controls were used:
without S9 mix:
"sodium azide (NaN3): TA 1535 strain,
"9-Aminoacridine (9AA): TA 1537and TA 97 strains,
"2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide : TA 98 and TA100 strains,
with S9 mix:
"2-Aminoanthracene: TA 1535, TA 1537, TA 98 and TA 100 strains
Results:
The total maximum dose of 5 mg per plate was selected as the highest dose of the experiment. The selected treatment-levels were 313, 625, 1250, 2500 and 5000 ug/plate for all strains with or without metabolic activation. The test item did not induce any significant increase in the number of revertants, in either experiment, in any of the five strains and no toxicity was observed.
Conclusion:
Under these experimental conditions, the test item 2,2-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
- 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
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. GLP guideline study (OECD 473)
- Justification for type of information:
- see 13.2 for attached read across rationale
- 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:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- mammalian cell line, other: Chinese hamster lung cells (CHL/IU)
- Details on mammalian cell type (if applicable):
- no data
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- -S9 mix (continuous treatment) : 0, 0.40, 0.80, 1.6 mg/L
-S9 mix (short-term treatment) : 0, 0.40, 0.80, 1.6 mg/L
+S9 mix (short-term treatment) : 0, 0.40, 0.80, 1.6 mg/L - Vehicle / solvent:
- 0.5% CMC (Carboxymethylcellulose) sodium solution
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: - S9 : Mitomycin C ; +S9 : Cyclophosphamide
- Details on test system and experimental conditions:
- Three doses of the test substance, together with the appropriate concurrent solvent and positive controls, were tested with and without metabolic activation for a 6H-period. Given the negative results from this protocol (short-treatment), a continous experiment was conducted for a 24H-period and a 48H-period witout activation.
- Evaluation criteria:
- Not precised
- Statistics:
- no
- Species / strain:
- mammalian cell line, other: Chinese hamster lung cells (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- See table (below)
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Under the experimental conditions described, the test substance 2,2’-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the mammalian chromosome aberration test. - Executive summary:
The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to cause structural chromosome aberrations in cultured Chinese hamster lung cells was evaluated according to OECD 473 guideline in compliance with the Principles of Good Laboratory Practice.
Methods:
The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with phenobarbital and 5,6-benzoflavone. The first experiment was performed for a short period of 6 hours with and without metabolic activation. The second one was a continuous treatment performed for a 24 hour and a 48 hour period but without metabolic activation. Each cultured cell was exposed to three dose-levels of the test item (two plates/dose-level).
The evaluation of the toxicity was performed on the basis of the observation of the increase in the number of cells with chromosome aberrations, in the number of polyploid cells or in the number of cells with endoreduplicated chromosomes.
The test item 2,2-AZOBIS(ISOBUTYRONITRILE) was dissolved in a 0.5% Carboxymethylcellulose sodium solution and the following positive controls were used:
- without S9 mix: Mytomycin C
- with S9 mix : Cyclophosphamide
Results:
The selected treatment-levels were 0.40, 0.80 and 1.6 mg/mL with or without metabolic activation. The test item did not induce any significant increase in the number of cells with chromosome aberrations, in the number of polyploid cells nor in the number of cells with endoreduplicated chromosomes in either experiment, and no toxicity was observed.
Conclusion:
Under these experimental conditions, the test item 2,2-AZOBIS(ISOBUTYRONITRILE) did not induce chromosome aberrations in cultured mammalian somatic cells.
- 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
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. Guideine study (OECD 476). No indication of GLP compliance, Restrictions: sample analyzed by MRI but purity not reported, the size of the colony was determined was not reported even for the solvent and positive controls.
- Justification for type of information:
- see 13.2 for attached read across rationale
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC).
- Cells were grown in Fisher's medium for leukemic cells of mice (Gibco, Grand Island, NY or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI.
- Cells were screened for the presence of mycoplasma after cryopreservation.
- New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat S9 mix. The microsomal enzyme fraction was prepared as described by Clive et al. (1979, Mutat. Res.59, 61-108). Liver S9 homogenate was prepared from male Sprague-Dawley rats that have been injected with Aroclor 1254
- Test concentrations with justification for top dose:
- 600, 700, 800, 900 and 1000 µg/mL
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with & without S9
- Positive control substance:
- other: Without S9 : ethylmethanesulphonate With S9 : 3-methylcholantrene (dimethylbenz[a]anthracene also used)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- The mutagenicity assay was performed according to Clive and Spector (Mutat Res., 31, 17-29).
- A total of 1.2*1E7 cells in duplicate cultures were exposed to the test chemical (as well as to solvent and positive control) for 4 hours at 37±1°C. After washing twice with growth medium, cells were maintained at 37±1°C for 48 hours in log-phase growth to allow recovery and mutant expression. Cells in the culture were adjusted to 3*1E5/mL at 24-hour intervals.
- Cells were cloned to 1*1E6 cells/plate for mutant selection and 200 cells/plate for viable count determinations in soft agar medium (contained Fisher's medium, 20% horse serum, 2mM sodium pyruvate, 0.02% pluronic F-68 and 0.23% granulated agar (BBL Inc., Cockeyville, MD)).
- Resistance to trifluorothymidine (TFT) was determined by adding TFT (3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37±1°C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, Dynatech) or Protocol colony counter (Synbiosis, Frederick, MD). Only colonies larger than 0.2 mm diameter (ca) were counted.
- Mutant frequencies were expressed as mutants per 1E6 surviving cells.
- The size of mutant mouse lymphoma colonies were also determined thanks to the Artek 982 colony counter/sizer or Protocol colony counter.The size range used was from 0.2 (ca) to 1.1 mm.
NUMBER AND SELECTION OF DOSES, CONTROLS USED
- The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.
- The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study. Cells at concentration of 6*1E5/mL were exposed for 4 hours to a range of concentrations from 0.0005 to 10000 µg/mL. The cells where then washed, resuspended in growth medium and incubated at 37°C (±1°C) for 48 hours. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding 0 - 90% cytotoxicity. For each assay, there were 2-4 solvent controls, a positive control of ethyl methylsulfonate at 4.7*1E-6M (or methyl methanesulfonate at 10-20 µg/mL) for the test without metabolic activation, and a positive control of 3-methylcholantrene at 1.86*1E-5M (or dimethylbenz[a]anthracene at 0.5-4 µg/mL) for the test with metabolic activation. - Evaluation criteria:
- For a test substance to be considered positive, it had to induce at least a doubling of the mutant frequency over the concurrent solvent-treated control value. This increase in the mean mutant frequency had to be accompanied by a dose response to increasing concentrations of the test substance. Only doses yielding total growth values of 10% or greater were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
- Statistics:
- No statistics were performed
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- See tables (below)
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Under the experimental conditions described, the test substance did not show any mutagenic activity in the mouse lymphoma assay. - Executive summary:
The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to induce gene mutation was evaluated in a mouse lymphoma mutagenicity assay using L5178Y TK+/- cells. The test method used was similar to OECD 476 guideline but purity of the product nor compliance with the Principles of Good Laboratory Practice were reported by the authors of the publication.
The test item was tested in an experiment with and without a metabolic activation system, the S9 mix. From the cytotoxicity results of the preliminary test, the total maximum dose of 1000 µg/mL was selected as the highest dose of the experiment. The selected treatment-levels were 600, 700, 800, 900 and 1000 µg/mL. Duplicate cultures of 1.2*1E7 cells were exposed to the test or control items for 4 hours at 37±1°C. At the end of the exposure period, cells were washed and maintained for 48 hours at 37±1°C to allow for the mutant expression, then cultured in appropriate medium with trifluorothymidine for 10 -12 days at 37±1°C to determine survival and to allow for the mutant selection (expression of the mutant phenotype). Mutant colonies and relative total growth were then scored.
The evaluation of the toxicity was performed on the basis of the observation of an increase in the number of mutant frequencies and/or a reduction in the growth rate (small colonies). The test item did not induce any significant increase in the mutation frequency and no toxicity was observed. Under these experimental conditions, the test item 2,2-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the in vitro Mammalian Cell Gene Mutation Test using L5178Y mouse lymphoma cells.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Only 4 strains instead of the 5 required by current guidelines were tested.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Remarks:
- Conducted according to guideline in effect at time of study conduct
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver (S-9 mix)
- Test concentrations with justification for top dose:
- 50 to 5000 µg
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- An aliquot (0.1 mL) of each concentration of test substance was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45°C, was then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL). Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of DMSO (0.1 ML) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates. Aliquots (0.1 mL) of a 10E-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell density of each culture. All plates were prepared in triplicate, allowed to solidify and incubated at 37°C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified. Results obtained with all strains were confirmed in a second, independent experiment.
- 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A solution of the test substance was prepared in DMSO at 25 mg/mL, and an aliquot of the solution (0.2 mL) was transferred to a sterile tube containing molten histidine-deficient top-agar (2.0 mL) maintained at 45°C. An additional aliquot (0.1 mL) of the test material solution was similarly transferred to another tube of molten top-agar (2.0 mL). Three serial 10-fold dilutions in molten top-agar were prepared from each of the above preparations, giving a series of 8 different concentrations of the test material from 2.5 µg to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA98 (0.1 mL) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, DMSP (0.2 mL) and culture, and top-agar and test material (0.1 mL) without bacterial culture. The plates were incubated for 37°C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material was shown by absence or thinning of the background lawn. No visible thinning of the background lawn of non-revertant cells was obtained. A top exposure level of 5 mg/plate was therefore selected for use in the main assay.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
It was concluded that the test item was devoid of mutagenic activity under the conditions of the test. - Executive summary:
The test substance was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays. Results obtained with all strains were confirmed in a second, independent experiment. The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of the test substance from 50 to 5000µg per plate, selected following a toxicity test, and included solvent (dimethyl suiphoxide) controls with and without S-9 mix. No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the levels tested, either in the presence or absence of S-9 mix. It was concluded that the test substance was devoid of mutagenic activity under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1993
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Short abstract from the 20th Annual meeting of the Japanese Society of Toxicological Sciences. No study details provided.
- Principles of method if other than guideline:
- The reverse mutation assay was conducted with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA in the absence and presence of metabolic activation system (S9 mix).
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Remarks:
- There were negative controls based on summary: did not increase the number of revertants compared with negative control values
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- other: see Remarks
- Remarks:
- from summary: 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
- Cytotoxicity / choice of top concentrations:
- not specified
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- other: see Remarks
- Remarks:
- 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
- Cytotoxicity / choice of top concentrations:
- not specified
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- other: see Remarks
- Remarks:
- 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
- Cytotoxicity / choice of top concentrations:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- other: see Remarks
- Remarks:
- 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
- Cytotoxicity / choice of top concentrations:
- not specified
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- other: see Remarks
- Remarks:
- 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
- Cytotoxicity / choice of top concentrations:
- not specified
- Conclusions:
- 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or
without metabolic activation. - Executive summary:
The reverse mutation assay was conducted with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA in the absence and presence of metabolic activation system (S9 mix).
2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
Referenceopen allclose all
Table 1 : Number of revertants per strain : 1st experiment
Table 2 : Number of revertants per strain : 2nd experiment
§Table 3 : Number of revertants per strain : Confirmation test
Table 4 : Number of revertants per strain: Additional experiment
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Table 1: Chromosome analysis of Chinese hamster cells continous treatment, without S9 mix.
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Table 2: Chromosome analysis of Chinese hamster cells short treatment, with and without S9 mix.
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Table 1: Mouse lymphoma test results both with and without activation
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Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Supporting in vivo micronucleus study on 13472-08-7 (supporting due to lack of reliability due to abstract only) is negative. Further testing not required as all in vitro endpoints are negative.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic and germ cell study: gene mutation
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The test substance was examined for mutagenic activity in Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, using pour-plate assays. Results obtained with all strains were confirmed in a second, independent experiment. The studies, which were conducted in the absence and presence of an activating system employed a range of levels of the test substance from 50 to 5000 µg per plate with and without a metabolic activation system. No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the levels tested. It was concluded that the test substance was devoid of mutagenic activity under the conditions of the test.
An Ames assay, chromosome aberration test, and mouse lymphoma assay with 2,2’-azobis(isobutyronitrile) were used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances share a common alerting group of azo which has the potential to drive outcomes in a battery of genotoxicity tests and as evidenced by other azo and azoxy compounds.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.
2,2’-Azobis(isobutyronitrile) was tested in an Ames assay with Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA, with and without a metabolic activation system, the S9 mix, and Salmonella typhimurium TA97 without metabolic activation. Different dose levels of the test material were tested on each strain of Salmonella. 2,2’-Azobis(isobutyronitrile) did not induce any significant increase in the number of revertants, in the Salmonella strains used and no toxicity was observed.In the chromosome aberration test with Chinese hamster lung cells, 2,2’-azobis(isobutyronitrile) did not induce any significant increase in the number of cells with chromosome aberrations, in the number of polyploid cells or in the number of cells with endoreduplicated chromosomes in the presence or absence of the metabolic activation system, and no toxicity was observed. The mouse lymphoma assay showed that 2,2’-azobis(isobutyronitrile) did not induce any significant increase in the mutation frequency and no toxicity was observed in the presence or absence of the metabolic activation system. Taken together the weight of evidence of these studies support the conclusion that the test substance is not considered genetically active.
Justification for selection of genetic toxicity endpoint
In addition to the Ames assay with the test substance, multiple OECG
guideline, GLP studies with the read-across chemical,
2,2’-azobis(isobutyronitrile) have been identified for this endpoint. In
addition the study selected above, Ames assay, in vitro chromosome
aberration assay, and mouse lymphoma assay with the read-across
chemical, 2,2’-azobis(isobutyronitrile) are pertinent to the hazard
conclusion for this endpoint.
Short description of key information:
The test substance was negative in the Ames assay. In addition, the
read-across chemical, 2,2’-azobis(isobutyronitrile), was negative in the
Ames assay, in vitro chromosome aberration assay, and mouse lymphoma
assay.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
The test substance was negative in the Ames assay. In addition, the read-across chemical, 2,2’-azobis(isobutyronitrile), was negative in Ames assay, in vitro chromosome aberration assay, and mouse lymphoma assay. Based on the weight of evidence provided, the test substance does not need to be classified for genetic toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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