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EC number: 217-589-7 | CAS number: 1897-52-5
- 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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 March 1992 to 2 October 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 993
- Report date:
- 1993
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (see field "Principles of method if other than guideline" for further information)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- (see field "Principles of method if other than guideline" for further information)
- Principles of method if other than guideline:
- Protocol Deviations:
- SOP TDS 203, 204, 205 and 207 were used in the experiments
- By mistake the temperature of the waterbath was not constantly measured during the overnight incubation (29 September 1992 to 30 September 1992) of the strains for the second mutation test. The temperature was measured once on 30 September 1992 at the end of the incubation period. At that moment the temperature of the waterbath was 37ºC.
- The test to determine the characteristics of the Salmonella typhimurium strains used in the first mutation study was done on 26 April 1991.
The above mentioned deviations are not considered to have interfered with the integrity of the study. - GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2,6-difluorobenzonitrile
- EC Number:
- 217-589-7
- EC Name:
- 2,6-difluorobenzonitrile
- Cas Number:
- 1897-52-5
- Molecular formula:
- C7H3F2N
- IUPAC Name:
- 2,6-difluorobenzonitrile
- Details on test material:
- - Identification: Diflubenil
- Physical state: Yellowish paste
Constituent 1
Method
- Target gene:
- S. typhimurium: Histidine synthesis
Species / strain
- Species / strain / cell type:
- other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Details on mammalian cell type (if applicable):
- - Type and identity of media: To tubes containing 2 ml histidine-deficient agar supplemented with a histidine/biotin mixture to support initial growth, maintained at a temperature of 52 ºC the following were added: 0.1 ml of an overnight growth culture of each of five strains, 0.1 ml of a solution of test material in DMSO and either 0.5 ml of the S9-mix or 0.5 ml cofactors mix. The components were mixed and rapidly poured onto a selective agar plate to gel.
- Properly maintained: yes. After solidification of the agar, the plates were turned upside down and incubated in the dark at 37ºC for 48-72 hours. S9-mix, cofactors mix and the test material solution were checked for sterility. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- 625, 1250, 2500 and 5000 µg/plate both in the presence and absence of metabolic activation.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- the vehicle of the test material (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: (details on positive control substance assigned is presented in Table 1 in the field "Any other information on materials and methods incl. tables")
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 - 72 hours
NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate
EVALUATION PROCEDURE: Following the total incubation period the plates were examined for the lack of microbial contamination. Each culture was also examined for the number of spontaneous revertants. Revertant colonies were counted automatically with an Artek 880 colony counter. - Evaluation criteria:
- A positive response in the assay system is considered to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the solvent control, together with evidence of a dose-response relationship. This response has to be greater than the laboratory solvent historical solvent control range. The positive response has to be reproducible in an independent experiment.
Results and discussion
Test results
- Species / strain:
- other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Tables 2 and 3 show the individual plate counts, the standard deviation and the mean number of revertant colonies of the five tester strains exposed to the test material together with the solvent control (DMSO) and positive controls.
In the first mutation study no increase in revertant colony counts was observed for any of the five tester strains, both in the presence and absence of the S9 -mix.
In the second mutation assay a two-fold increase in colony counts was observed with the test material at concentrations of 2500 µg/plate and 5000 µg/plate with strain TA 1535 in the absence of the S9 -mix only. This increase in the number of revertants fell within the laboratory historical solvent control range (10 ±4). All other strains showed no increase of revertant colony counts, compared to the solvent control, neither in the presence or absence of the S9 -mix.
Table 2: Salmonella/Microsome Mutation assay 1
Dose (µg/ plate) | TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1538 | ||||||
+S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | ||
0 | 31 | 38 | 94 | 85 | 4 | 9 | 8 | 20 | 18 | 24 | |
31 | 52 | 83 | 73 | 5 | 13 | 14 | 23 | 20 | 17 | ||
32 | 32 | 91 | 87 | 8 | 12 | 20 | 14 | NB | 19 | ||
Mean | 31 | 41 | 89 | 82 | 6 | 11 | 14 | 19 | 18; 20 | 20 | |
SD | 1 | 10 | 6 | 8 | 2 | 2 | 6 | 5 | 4 | ||
625 | 24 | 38 | 87 | 92 | 5 | 5 | 15 | 12 | 20 | 22 | |
20 | 35 | 66 | 100 | 5 | 7 | 6 | 16 | 18 | 31 | ||
16 | 31 | 87 | 97 | 7 | 2 | 10 | 22 | 30 | 20 | ||
Mean | 20 | 35 | 80 | 96 | 6 | 5 | 10 | 17 | 23 | 24 | |
SD | 4 | 4 | 12 | 4 | 1 | 3 | 5 | 5 | 6 | 6 | |
1250 | 31 | 34 | 95 | 92 | 5 | 12 | 11 | 15 | 18 | 20 | |
24 | 47 | 69 | 100 | 7 | 6 | 13 | 16 | 17 | 24 | ||
17 | 35 | 81 | 92 | 7 | 8 | 14 | 19 | 20 | 31 | ||
Mean | 24 | 39 | 82 | 95 | 6 | 9 | 13 | 17 | 18 | 25 | |
SD | 7 | 7 | 13 | 5 | 1 | 3 | 2 | 2 | 2 | 6 | |
2500 | 16 | 31 | 91 | 83 | 13 | 11 | 11 | 13 | 31 | 18 | |
22 | 35 | 66 | 100 | 6 | 16 | 16 | 18 | 30 | 24 | ||
22 | 33 | 67 | 86 | 9 | 10 | 8 | 16 | 33 | 15 | ||
Mean | 20 | 33 | 75 | 90 | 9 | 12 | 12 | 16 | 31 | 19 | |
SD | 3 | 2 | 14 | 9 | 4 | 3 | 4 | 3 | 2 | 5 | |
5000 | 23 | 45 | 56 | 69 | 5 | 6 | 8 | 16 | 16* | 21* | |
21 | 32 | 34 | 81 | 4 | 2 | 10 | 12* | 30 | 22 | ||
22 | 38 | 62 | 45 | 7 | 6 | 13 | 14* | 18* | 32 | ||
Mean | 22 | 38 | 50 | 65 | 5 | 5 | 10 | 14* | 21 | 25 | |
SD | 1 | 7 | 14 | 18 | 2 | 2 | 3 | 2 | 8 | 6 | |
Positive Control | 2122 | 1952 | 217 | 919 | 327 | 35 | 240 | 227 | 2330 | 1757 | |
2183 | 1941 | 279 | 890 | 294 | 32 | 211 | 183 | 1882 | 1727 | ||
2334 | 2364 | 217 | 979 | 290 | 30 | 273 | 231 | 2647 | 1824 | ||
Mean | 2213 | 2086 | 238 | 929 | 304 | 32 | 241 | 214 | 2286 | 1769 | |
SD | 109 | 241 | 36 | 45 | 20 | 3 | 31 | 27 | 384 | 50 | |
Mean: Average number of revertants per plate
SD: standard deviation
S9: liver homogenate from rats treated with Aroclor
* Toxic
NB: no background lawn (and therefore no colony forming)
Table 3: Salmonella/Microsome Mutation assay 2
Dose (µg/ plate) | TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1538 | ||||||
+S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | ||
0 | 18 | 30 | 90 | 93 | 6 | 5 | 7 | 6 | 64 | 63 | |
18 | 31 | 80 | 85 | 7 | 9 | 2 | 4 | 69 | 57 | ||
27 | 41 | 93 | 85 | 5 | 9 | 9 | 9 | 51 | 54 | ||
Mean | 21 | 34 | 88 | 88 | 6 | 8 | 6 | 6 | 62 | 58 | |
SD | 5 | 6 | 7 | 5 | 1 | 2 | 4 | 3 | 9 | 5 | |
625 | 27 | 26 | 99 | 85 | 9 | 4 | 4 | 5 | 84 | 87 | |
26 | 28 | 98 | 76 | 9 | 6 | 6 | 9 | 66 | 51 | ||
27 | 30 | 79 | 66 | 10 | 7 | 5 | 2 | 68 | 50 | ||
Mean | 27 | 28 | 92 | 76 | 9 | 6 | 5 | 5 | 73 | 63 | |
SD | 1 | 2 | 11 | 10 | 1 | 2 | 1 | 4 | 10 | 21 | |
1250 | 27 | 33 | 57 | 93 | 5 | 9 | 6 | 4 | 78 | 55 | |
24 | 17 | 56 | 78 | 12 | 11 | 6 | 8 | 85 | 41 | ||
31 | 20 | 69 | 85 | 10 | 4 | 6 | 4 | 69 | 39 | ||
Mean | 27 | 23 | 62 | 85 | 9 | 8 | 6 | 5 | 77 | 45 | |
SD | 4 | 9 | 7 | 8 | 4 | 4 | 0 | 2 | 8 | 9 | |
2500 | 29 | 18 | 91 | 77 | 16 | 8 | 5 | 4 | 32 | 27 | |
18 | 29 | 76 | 99 | 16 | 8 | 7 | 8 | 88 | 7 | ||
29 | 25 | 84 | 88 | 10 | 5 | 9 | 5 | 29 | 7 | ||
Mean | 25 | 24 | 84 | 88 | 14 | 7 | 7 | 6 | 50 | 14 | |
SD | 6 | 6 | 8 | 11 | 3 | 2 | 2 | 2 | 33 | 12 | |
5000 | 20 | 21 | 80 | 73 | 9 | 5 | 3 | 6 | 48 | 0 | |
29 | 18 | 74 | 78 | 14 | 9 | 3 | 5 | 32 | 0 | ||
14 | 27 | 52 | 49 | 15 | 3 | 4 | 7 | 44 | 0 | ||
Mean | 21 | 22 | 69 | 67 | 13 | 6 | 3 | 6 | 41 | 0 | |
SD | 8 | 5 | 15 | 16 | 3 | 3 | 1 | 1 | 8 | 0 | |
Positive Control | 681 | 1831 | 608 | 1390 | 726 | 112 | 265 | 170 | 1418 | 103* | |
391 | 617 | 637 | 1318 | 640 | 104 | 299 | 166 | 1451 | 554 | ||
1467 | 1703 | 581 | 1214 | 665 | 111 | 298 | 155 | 2426 | 757 | ||
Mean | 846 | 1384 | 609 | 1307 | 677 | 109 | 287 | 164 | 1765 | 554 | |
SD | 557 | 667 | 28 | 88 | 44 | 4 | 19 | 8 | 573 | 757 |
Mean: Average number of revertants per plate
SD: standard deviation
S9: liver homogenate from rats treated with Aroclor
* Toxic
NB: no background lawn (and therefore no colony forming)
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
At 2500 and 5000 µg/plate, the test material showed a two-fold increase of the number of revertants in strain TA 1535 in the absence of the S9-mix. Since this increase fell within the laboratory historical range of background revertant counts and because the effect was not reproducible, it was concluded that the test material showed no evidence of mutagenic potential both in the presence and in the absence of the S9-mix in the AMES bacterial system at the dose levels used.
Under the conditions of the study, the test material gave a negative (i.e. non-mutagenic) response in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 both in the presence and absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and for classification and labelling purposes. - Executive summary:
The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standard guidelines OECD 471 and EU method B.14. Five strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). In two separate assays the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the presence or in the absence of metabolic activation. It was therefore concluded that the test material showed no evidence of mutagenic potential both in the presence and in the absence of metabolic activation at the dose levels used.
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