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EC number: 257-768-7 | CAS number: 52236-73-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From September 29th to October 26th, 1992
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Justification for type of information:
- Justification for Read Across is reported in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 992
- Report date:
- 1992
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted May 26, 1983
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Similar Substance 02
- IUPAC Name:
- Similar Substance 02
Constituent 1
Method
- Target gene:
- Salmonella typhimurium histidine (his) reversion system.
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- - Supplier: bacterial strains were obtained from Dr. Heinz Träger, Knoll AG, D-6700 Ludwigshafen, F.R.G.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.
- Incubation: the bacterial culture was incubated in a shaking water bath for 10 hours at 37° C.
- Regular checking: regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix (Rat liver)
- Test concentrations with justification for top dose:
- Pre-experiment for toxicity: 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/ml
Main study (two independent experiments): 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/ml - Vehicle / solvent:
- - Vehicle test substance was dissolved in aqua bidest.
- Justification for choice of vehicle: the solvent was chosen because of its solubility properties.
Controls
- Untreated negative controls:
- yes
- Remarks:
- concurrent untreated
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION
EXPERIMENT I: PLATE INCORPORATION TEST
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control); 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation); 100 µl bacteria suspension (cf. test system, pre-culture of the strains); 2000 µl overlay agar.
EXPERIMENT II: PRE-INCUBATION TEST
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix /S9 mix substitution buffer and 100 µl bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
NUMBER OF REPLICATIONS: each concentration, including the controls, was tested in triplicate.
DATA RECORDING
The colonies were counted using the AUTOCOUNT. The counter was connected to a compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.
PRE-EXPERIMENT FOR TOXICITY
- Strains: to evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and TA 100.
- Concentrations: 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/ml
- Replicates: 3 plates per concentration.
- Experimental conditions: the same as described for the experiment I (plate incorporation test).
- Evaluation: toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.
MAIN TEST COCNENTRATIONS
- Concentrations: the maximum concentration was 5000.0 µg/plate; the concentration range included two logarithmic decades.
- Spacing factor: six adequately spaced concentrations were tested.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9 Preparation: the S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU (weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1+3 in KCl was centrifuged cold at 9000 g for 10 minutes.
- Storage of stock solution: stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The protein concentration in the S9 preparation was 33.6 mg/ml.
- S9 Mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 1.5:8.5. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- Storage: during the experiment the S9 mix was stored in an ice bath.
- Preparation: the S9 mix preparation was performed according to Ames et al.
ACCEPTANCE CRITERIA
The generally accepted conditions for the evaluation of the results are:
- Corresponding background growth on both negative control and test plates
- Normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies (the following values refer to the negative control without metabolic activation (+) The range of strain TA 102 was determined from testing laboratory historical control datas)
TA 1535: 3 - 37
TA 1537: 4 - 31
TA 1538: 12 - 37
TA 98: 15 - 60
TA 100: 75 - 200
TA 102 (+): 120 - 300 - Evaluation criteria:
- A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: a test article is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate; also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not. - Statistics:
- No evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at the test performance time.
Results and discussion
Test results
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No toxic effects occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.
A slight increase in revertant colony numbers (factor 1.5) was observed in strain TA 100 with metabolic activation at the highest investigated dose in experiment II (pre-incubation test). This effect is considered not to be biologically relevant. It is due to the relatively low level of the corresponding solvent control. Additionally the factor of 2.0 which is recommended for a mutagenic response in strain TA 100 could not be reached.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.
PRE-EXPERIMENT FOR TOXICITY
The pre-study was performed with strains TA 98 and TA 100.
The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.
Any other information on results incl. tables
SUMMARY OF RESULTS
Revertants/plate mean from three plates, without S9 mix
Dose µg/plate | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 102 | |||||
I | II | I | II | I | II | I | II | I | II | |
Neg. contr. | 11 | 24 | 8 | 12 | 17 | 33 | 91 | 74 | 144 | 206 |
Solv. contr. | 12 | 25 | 6 | 12 | 26 | 28 | 90 | 77 | 177 | 208 |
33.3 | 8 | 29 | 9 | 13 | 15 | 28 | 100 | 68 | 190 | 208 |
100.0 | 9 | 30 | 8 | 13 | 15 | 29 | 98 | 72 | 188 | 175 |
333.3 | 9 | 23 | 9 | 11 | 20 | 25 | 82 | 72 | 152 | 201 |
1000.0 | 9 | 29 | 8 | 13 | 15 | 28 | 89 | 79 | 202 | 225 |
2500.0 | 9 | 28 | 8 | 14 | 26 | 29 | 85 | 76 | 167 | 207 |
5000.0 | 10 | 27 | 8 | 12 | 19 | 29 | 108 | 81 | 184 | 199 |
Positive controls | ||||||||||
Sodium azide (10 µg/plate) |
856 |
655 |
1018 |
1105 |
||||||
4-Nitro-o-phenylene-diamine (50 µg/plate) |
647 |
302 |
1972 |
2280 |
1116 |
1233 |
Revertants/plate mean from three plates, with S9 mix
Dose µg/plate | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 102 | |||||
I | II | I | II | I | II | I | II | I | II | |
Neg. contr. | 12 | 22 | 11 | 19 | 21 | 46 | 98 | 95 | 225 | 279 |
Solv. contr. | 13 | 25 | 8 | 24 | 22 | 49 | 104 | 85 | 296 | 310 |
33.3 | 10 | 23 | 9 | 23 | 29 | 49 | 93 | 75 | 278 | 263 |
100.0 | 14 | 22 | 9 | 24 | 22 | 47 | 112 | 90 | 280 | 273 |
333.3 | 11 | 26 | 9 | 21 | 19 | 49 | 115 | 96 | 265 | 315 |
1000.0 | 14 | 24 | 8 | 22 | 21 | 45 | 113 | 100 | 332 | 366 |
2500.0 | 12 | 23 | 9 | 23 | 19 | 47 | 116 | 112 | 328 | 331 |
5000.0 | 10 | 27 | 9 | 22 | 24 | 44 | 135 | 124 | 320 | 381 |
Positive control | ||||||||||
2-Aminoanthracene (2.5 µg/plate) | 416 | 422 | 355 | 351 | 937 | 668 | 898 | 789 | 1515 | 966 |
PRE-EXPERIMENT FOR TOXICITY
Test groups |
Concentration per plate µg |
Revertants per plate |
|||
- |
+ |
- |
+ |
||
Negative control |
- |
17 |
21 |
91 |
98 |
Solvent control |
- |
26 |
22 |
90 |
104 |
4-NOPD |
50 |
1972 |
/ |
/ |
/ |
Sodium azide |
10 |
/ |
/ |
1018 |
/ |
2-aminoanthracene |
2.5 |
/ |
937 |
/ |
898 |
Test article |
3.3 |
14 |
25 |
93 |
107 |
10 |
22 |
18 |
88 |
124 |
|
33.3 |
15 |
29 |
100 |
93 |
|
100 |
15 |
22 |
98 | 112 | |
333.3 | 20 | 19 | 82 | 115 | |
1000 | 15 | 21 | 89 | 113 | |
2500 | 26 | 19 | 85 | 116 | |
5000 | 19 | 24 | 108 | 135 |
* -: without S9 mix; +: with S9 mix
/ not performed
Applicant's summary and conclusion
- Conclusions:
- Dring the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic.
- Executive summary:
Method
The study was performed to investigate the potential of test material to induce gene mutations, according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and. TA 102.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate.
Results
No toxic effects occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used.
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
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