Registration Dossier
Registration Dossier
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EC number: 201-132-3 | CAS number: 78-67-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Four separate AMES studies were used to investigate the potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to induce reverse mutation in Salmonella typhimurium, the studies were run according to or used similar method to OECD 471 guideline.
A chromosome aberration test was run according to OECD Guideline 473 and the Japanese Guideline for screening mutagenicity testing of chemical.
In addition to the above in vitro tests a mouse lymphoma assay was run on AZDN according to OECD Guideline 476 and a second according to OECD 490 which were negative (Labcorp, 2022)
Endpoint Conclusion: No adverse effect observed (negative)
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study (OECD 473)
- 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 (migrated information):
negative
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. According to the criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations), the test item 2,2'-AZOBIS(ISOBUTYRONITRILE) is considered as non-mutagenic and is not classified.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start date: 23 April 2021
Experimental completion date: 17 May 2021 - Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Method B67 of Commission Regulation (EC) No. 440/2008
- Version / remarks:
- 26 September 2019.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 Microsomal Enzyme Fraction was purchased from Moltox, Lot no 4370 with the expiry date of 24 November 2022, was used in this study.
The protein content was adjusted to approximately 20 mg/ml prior to use.
The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5
mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test. - Test concentrations with justification for top dose:
- The concentrations used in the preliminary toxicity test were 0, 3.21, 6.41, 12.83, 25.66, 51.32, 102.63, 205.26, 410.52, and 821.04 µg/mL.
For the Main test the exposures were performed in duplicate (A + B) at eight concentrations of the test item (0, 3.21, 6.41, 12.83, 25.66, 51.32, 102.63, 205.26, 410.52, and 821.04 µg/mL in both of the exposure groups), solvent and positive controls. - Vehicle / solvent:
- DMSO.
The test item was insoluble in culture medium at 16.4 mg/mL, and DMSO at 164.2 mg/mL.
A solution suitable for dosing was achieved at 82.1 mg/mL, the maximum practical concentration, in DMSO. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier. Master stocks of cells were tested and found to be free of mycoplasma
Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Test Item Preparation
The molecular weight of the test item was 164.208 and therefore the maximum concentration in the solubility check was set at 1642.08 µg/mL, the 10 mM limit concentration. However, the test item was insoluble in culture medium at 16.4 mg/mL, and DMSO at 164.2 mg/mL. A solution suitable for dosing was achieved at 82.1 mg/mL, the maximum practical concentration, in DMSO. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 1 x 107 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9). The maximum concentration used was based on precipitate observed in the solubility. The concentrations used in the preliminary toxicity test were 0, 3.21, 6.41, 12.83, 25.66, 51.32, 102.63, 205.26, 410.52, and 821.04 µg/mL. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL in R20 medium. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item concentrations for the mutagenicity experiments. Maximum concentrations were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper concentration may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating concentrations will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA
Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate (A + B) at eight concentrations of the test item (0, 3.21, 6.41, 12.83, 25.66, 51.32, 102.63, 205.26, 410.52, and 821.04 µg/mL in both of the exposure groups), solvent and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL.
The exposure vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
Measurement of Survival, Cloning Efficiency and Mutant Frequency
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO² in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the cloning efficiency (%V) data, a Relative Total Growth (RTG) value.
Plate Scoring
96 well plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualisation of the mutant colonies, particularly the small colonies. - Evaluation criteria:
- Please see " Any other information on materials and methods incl. methods" .
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- A summary of the results from the test is presented in Table 1 (Please see the attachment).
The results of the 96-well plate counts and their analysis are presented in Table 2 and Table 4 (Please see the attachment)
.
There was no evidence of any toxicity, in either of the exposure groups, following exposure to the test item, as indicated by the %RSG and RTG values (Table 2 and Table 4, please see the attachment). There was also no evidence of any marked reductions in cloning efficiency (%V) in either of the exposure groups, therefore indicating that residual toxicity had not occurred (Table 2 and Table 4 please see the attachment). Test item precipitate was observed at the maximum practical dose level of 821.04 µg/mL in both of the exposure groups. Acceptable levels of toxicity were seen with the positive control substances (Table 2 and Table 4 please see the attachment).
The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the maximum practical concentration, which was also the precipitating concentration, in both of the exposure groups, and at least four analysable concentrations, as recommended by the OECD 490 guideline. The results observed were considered to fulfill the criteria for a clearly negative outcome. - Remarks on result:
- other: AZDN did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic.t
- Conclusions:
- The test item, AZDN, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.
- Executive summary:
Introduction
This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Results
The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the maximum practical concentration, which was also the precipitating concentration, in both of the exposure groups, and at least four analysable concentrations, as recommended by the OECD 490 guideline. The results observed were considered to fulfill the criteria for a clearly negative outcome.
Conclusion
The test item, AZDN, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay in which all acceptability criteria were met.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- not stated
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study with acceptable restriction. No indication of GLP compliance. Restrictions : sample analyzed by MRI but purity not reported, positive controls used in the assay but identity not reported
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat S9 mix. The microsomal enzyme fraction was prepared as described by Ames et al. (1975, Mutat. Res.31, 347-364). Liver S9 homogenate was prepared from male Sprague-Dawley rats that have been injected with Aroclor 1254 at 500 mg/kg body weight
- Test concentrations with justification for top dose:
- 0, 100, 333, 1000, 3333 and 10000 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:
- not specified
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- The test was performed according to the direct plate incorporation method. For testing in the absence of S9 mix, 100 µl of the tester strain and 50 µl of the solvent or the test substance were added to 2.5 ml of molten selective top agar at 45+/- 2°C. When S9 was used, 0.5 ml of S9 mix , 50 µl of tester strain and 50 µl of solvent or test substance were added to 2.0 ml of molten selective top agar at 45+/- 2°C. After rapid homogenization, the mixture was overlaid onto a petrie plate containing minimum medium. The plates were incubated for 48h at 37+/- 2°C.
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 doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA 100. Ten dose levels of the test substance, one plate per dose, were tested in both the presence and the absence of S9 mix. If no toxicity was observed, a total maximum dose of 10 mg of test substance per plate was used. - Evaluation criteria:
- For a test substance to be considered positive, it had to induce at least a doubling (TA 98, TA 100 and TA 1535) in the mean number of revertants per plate of a least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance. If the study showed a dose response with a less than 3-fold increase on TA 1537 or TA 1538, the response had to be confirmed in a repeat experiment.
- Statistics:
- No statistics were performed. For each tester strain, mean number of revertant colonies per plate with the corresponding standard deviation were given.
- Species / strain:
- S. typhimurium, other: TA98, TA 100, TA 1535, TA 1537 and TA 1538
- 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:
- The number of revertants for positive controls in each Salmonella strain was provided but the identity of these controls was not stated.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
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 - Executive summary:
The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to induce gene mutation was evaluated in a bacterial reverse mutation test using Salmonella typhimurium. The test method used was similar to OECD 471 guideline but compliance with the Principles of Good Laboratory Practice was not 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, performed according to the direct plate incorporation method. Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were used and each strain was exposed to at least five dose-levels of the test item (three plates/dose-level).
Since the test item was not toxic in a preliminary test, the total maximum dose of 10 mg per plate was selected as the highest dose of the experiment. The selected treatment-levels were 100, 333, 1000, 3333 and 10000 µg/plate for all strains with or without metabolic activation.
After 48 hours of incubation at 37°C, the revertant colonies were scored.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 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.
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.
Referenceopen allclose all
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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Mean number and standard deviation of revertants per strain (n =3)
|
TA 98 |
TA 98 |
TA100 |
TA 100 |
TA 1535 |
TA1535 |
TA 1537 |
TA 1537 |
TA 1538 |
TA1538 |
|
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
DMSO |
28 ± 2 |
26 ± 5 |
159 ± 17 |
191 ± 11 |
6 ± 3 |
10 ± 5 |
6 ± 1 |
5 ± 2 |
6 ± 3 |
7 ± 2 |
100ug |
25 ± 3 |
32 ± 4 |
171 ± 6 |
178 ± 16 |
9 ± 3 |
8 ± 2 |
8 ± 3 |
6 ± 2 |
6 ± 3 |
9 ± 2 |
333ug |
25 ± 3 |
30 ± 7 |
157 ± 7 |
174 ± 28 |
10 ± 5 |
10 ± 3 |
8 ± 2 |
5 ± 3 |
6 ± 1 |
13 ± 4 |
1000ug |
30 ± 4 |
28 ± 5 |
169 ± 19 |
172 ± 18 |
9 ± 2 |
10 ± 2 |
5 ± 2 |
5 ± 1 |
12 ± 4 |
11 ± 2 |
3333ug |
32 ± 3 |
28 ± 2 |
164 ± 17 |
173 ± 13 |
9 ± 2 |
8 ± 1 |
3 ± 2 |
5 ± 3 |
15 ± 2 |
10 ± 2 |
10000ug |
22 ± 6 |
16 ± 1 |
93 ± 55 |
148 ± 22 |
6 ± 2 |
5 ± 2 |
7 ± 1 |
7 ± 1 |
19 ± 5 |
11 ± 5 |
Positive controls |
166 ± 20 |
1978 ± 211 |
591 ± 14 |
1727 ±123 |
295 ± 17 |
80 ± 14 |
581±139 |
148 ±14 |
458 ±13 |
1529 ±112 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The AMES tests used five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 97 or TA 1538,
with and without a metabolic activation system, the S9 mix. Different dose levels of the test material were tested on each strain of Salmonella.
AZDN 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, AZDN 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 the presence or absence of the metabolic activation system, and no toxicity was observed.
The Mouse lymphoma assay showed that AZDN 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
Justification for classification or non-classification
According to EU regulation (EC) No 1272/2008 (CLP) , AZDN was unclassified for mutagenicity endpoint.
AZDN was not mutagenic in any of the above in vitro models used : Ames test, mouse lymphoma test, and in mammalian chromosomic aberrations test.
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
