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EC number: 309-269-1 | CAS number: 100208-66-0
- 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 cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 July- 22 November, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 29th July, 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 14 February 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine
- EC Number:
- 290-904-3
- EC Name:
- Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine
- Cas Number:
- 90268-98-7
- Molecular formula:
- Molecular formula is not available
- IUPAC Name:
- Reaction product of aniline, 4-nitrobenzenamine, p-phenylenediamine and p-toluidine with sodium polysulfide
- Test material form:
- solid: particulate/powder
- Details on test material:
- Test item: Yellow 22
Appearance: ocher clay, solid
CAS No. 90268-98-7
EC No. 290-904-3
Storage: room temperature
Constituent 1
- Specific details on test material used for the study:
- Date of production: 22.05.2015
Expiration date: 22.05.2020
Method
- Target gene:
- Chromatid and chromosome type aberrations in metaphase cells
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with
L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.
- Cytokinesis block (if used):
- Cell cultures were treated with colchicine (0.2 µg/mL) 2.5 hours prior to harvesting
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 40.3 and 33.8 mg/mL.
- Test concentrations with justification for top dose:
- Experiment A (3/20h): 15.6,1 31.3, 62.5, 125 and 1802 μg/mL test item with and without S9 mix
Experiment B (20/20h and 20/28h): 3.9, 1 7.8, 15.6, 31.3 and 452 μg/mL test item without S9 mix
Experiment B (3/28h): 15.6, 1 31.3, 62.5, 125 and 180 μg/mL test item with S9 mix - Vehicle / solvent:
- Dimethyl sulfoxide DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- other: DME (Dulbecco’s Modified Eagle’s) medium
- Details on test system and experimental conditions:
- The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
Mammalian Microsomal Fraction S9 Mix
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 40.3 and 33.8 mg/mL.
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.
The S9 Mix (with Rat Liver S9)
The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= β-Nicotinamide Adenine Dinucleotide Phosphate
Before adding to the culture medium the S9 mix was kept in an ice bath. - Rationale for test conditions:
- Acceptability of the Assay
The chromosome aberration assay is considered acceptable because it meets the following criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
– concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
– cell proliferation in the solvent control is adequate,
– adequate number of cells and concentrations are analyzable,
– all requested experimental conditions were tested unless one resulted in a positive result
– the criteria for the selection of top concentration are fulfilled. - Evaluation criteria:
- Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.
Interpretation of Results
Providing that all acceptability criteria are fulfilled, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response. - Statistics:
- For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Solubility and Dose Selection
A homogeneous suspension of the test item was obtained in DMSO up to a concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested.
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with the test item are presented in Table 2A - 2C.
Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:
Experiment A with 3/20 h treatment/sampling time
without: 15.6,1 31.3, 62.5, 125 and 1802 *g/mL test item
with S9 mix: 15.6,1 31.3, 62.5, 125 and 180 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 3.9, 1 7.8, 15.6, 31.3 and 452 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 3.9, 1 7.8, 15.6, 31.3 and 452 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15.6, 1 31.3, 62.5, 125 and 180 *g/mL test item
1These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next higher concentration and sufficient number of concentrations.
2These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations.
All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.
Chromosome Aberration Assay
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item (Tables 13 and 14).
In both experiments, clear cytotoxicity of about 50% was observed after test item treatment in the absence and presence of metabolic activation.
No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation
In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values (5-6 aberrant cells excluding
gaps/150 cells) were slightly above the 95% control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.
The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 *L/mL) and Cyclophosphamide (5 *g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data .Thus, the study is considered valid. - Remarks on result:
- other: In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
- Remarks:
- test item is considered as being non-clastogenic in this system
Any other information on results incl. tables
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
3-hour treatment without and with S9 mix / 20-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
1850000 |
1900000 |
1918750 |
- |
- |
- |
- |
B |
– |
2050000 |
1950000 |
|||||
- |
C |
– |
2000000 |
1800000 |
|||||
- |
D |
– |
1850000 |
1950000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
6700000 |
7000000 |
6862500 |
4943750 |
100,00 |
0,00 |
- |
B |
– |
6850000 |
6900000 |
|||||
test item |
31.3 |
A |
– |
6800000 |
6800000 |
6800000 |
4881250 |
98,74 |
1,26 |
62.5 |
A |
– |
5400000 |
5150000 |
5275000 |
3356250 |
67,89 |
32,11 |
|
125 |
A |
– |
4300000 |
4400000 |
4350000 |
2431250 |
49,18 |
50,82 |
|
250 |
A |
– |
3150000 |
3000000 |
3075000 |
1156250 |
23,39 |
76,61 |
|
500 |
A |
– |
2500000 |
2450000 |
2475000 |
556250 |
11,25 |
88,75 |
|
EMS 1 µL/mL |
A |
– |
4600000 |
4600000 |
4600000 |
2681250 |
54,24 |
45,76 |
|
Solvent control (DMSO) |
- |
A |
+ |
6600000 |
6850000 |
6700000 |
4781250 |
100,00 |
0,00 |
- |
B |
+ |
6650000 |
6700000 |
|||||
test item |
31.3 |
A |
+ |
6600000 |
6600000 |
6600000 |
4681250 |
97,91 |
2,09 |
62.5 |
A |
+ |
5800000 |
6100000 |
5950000 |
4031250 |
84,31 |
15,69 |
|
125 |
A |
+ |
4400000 |
4200000 |
4300000 |
2381250 |
49,80 |
50,20 |
|
250 |
A |
+ |
3600000 |
3700000 |
3650000 |
1731250 |
36,21 |
63,79 |
|
500 |
A |
+ |
3250000 |
3250000 |
3250000 |
1331250 |
27,84 |
72,16 |
|
Cycl. 5µg/mL |
A |
+ |
4350000 |
4500000 |
4425000 |
2506250 |
52,42 |
47,58 |
RICC=Relative Increase in Cell Counts
Cytotoxicity= 100-RICC
DME: (Dulbecco’s Modified Eagle’s)medium
EMS: Ethyl methanesulfonate (EMS)
Cycl: Cyclophosphamide monohydrate
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
20-hour treatment without S9 mix / 20-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
1850000 |
1900000 |
1918750 |
- |
- |
- |
- |
B |
– |
2050000 |
1950000 |
|||||
- |
C |
– |
2000000 |
1800000 |
|||||
- |
D |
– |
1850000 |
1950000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
5400000 |
5500000 |
5487500 |
3568750 |
100,00 |
0,00 |
- |
B |
– |
5600000 |
5450000 |
|||||
test item |
7.8 |
A |
– |
5500000 |
5450000 |
5475000 |
3556250 |
99,65 |
0,35 |
15.7 |
|
|
4250000 |
4500000 |
4375000 |
2456250 |
68,83 |
31,17 |
|
31.3 |
A |
– |
3600000 |
3700000 |
3650000 |
1731250 |
48,51 |
51,49 |
|
62.5 |
A |
– |
3000000 |
3000000 |
3000000 |
1081250 |
30,30 |
69,70 |
|
125 |
A |
– |
1800000 |
1700000 |
1750000 |
-168750* |
-4,73** |
104,73*** |
|
250 |
A |
– |
1250000 |
1150000 |
1200000 |
-718750* |
-20,14** |
120,14*** |
|
500 |
A |
– |
900000 |
1000000 |
950000 |
-968750* |
-27,15** |
127,15*** |
|
EMS 1 µL/mL |
A |
– |
3600000 |
3550000 |
3575000 |
1656250* |
46,41** |
53,59*** |
RICC=Relative Increase in Cell Counts
Cytotoxicity= 100-RICC
DME: (Dulbecco’s Modified Eagle’s)medium
EMS: Ethyl methanesulfonate (EMS)
*: cell number decrease,
**: zero RICC value,
***:100% cytotoxicity
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
1850000 |
1900000 |
1918750 |
- |
- |
- |
- |
B |
– |
2050000 |
1950000 |
|||||
- |
C |
– |
2000000 |
1800000 |
|||||
- |
D |
– |
1850000 |
1950000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
8600000 |
8350000 |
8450000 |
6531250 |
100,00 |
0,00 |
- |
B |
– |
8500000 |
8350000 |
|||||
test item |
7.8 |
A |
– |
8200000 |
8300000 |
8250000 |
6331250 |
96,94 |
3,06 |
15.7 |
A |
– |
6950000 |
6750000 |
6850000 |
4931250 |
75,50 |
24,50 |
|
31.3 |
A |
– |
5150000 |
5000000 |
5075000 |
3156250 |
48,33 |
51,67 |
|
62.5 |
A |
– |
3850000 |
3600000 |
3725000 |
1806250 |
27,66 |
72,34 |
|
125 |
A |
– |
1900000 |
1850000 |
1875000 |
-43750* |
-0,67** |
100,67*** |
|
250 |
A |
– |
1300000 |
1300000 |
1300000 |
-618750* |
-9,47** |
109,47*** |
|
500 |
A |
– |
700000 |
600000 |
650000 |
-1268750* |
-19,43** |
119,43*** |
|
EMS 1 µL/mL |
A |
– |
4900000 |
5050000 |
4975000 |
3056250* |
46,79** |
53,21*** |
|
Solvent control (DMSO) |
- |
A |
+ |
8800000 |
8950000 |
8762500 |
6843750 |
100,00 |
0,00 |
- |
B |
+ |
8600000 |
8700000 |
|||||
test item |
31.3 |
A |
+ |
8800000 |
8550000 |
8675000 |
6756250 |
98,72 |
1,28 |
62.5 |
A |
+ |
7600000 |
7500000 |
7550000 |
5631250 |
82,28 |
17,72 |
|
125 |
A |
+ |
5200000 |
5300000 |
5250000 |
3331250 |
48,68 |
51,32 |
|
250 |
A |
+ |
4400000 |
4600000 |
4500000 |
2581250 |
37,72 |
62,28 |
|
500 |
A |
+ |
3900000 |
4000000 |
3950000 |
2031250 |
29,68 |
70,32 |
|
Cycl. 5µg/mL |
A |
+ |
5150000 |
4950000 |
5050000 |
3131250 |
45,75 |
54,25 |
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT A
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
incl. gaps |
excl. gaps |
||||
Negative (Solvent) control |
- |
3 h |
20 h |
7 |
3 |
test item |
|||||
31.3 µg/mL |
- |
3 h |
20 h |
12 |
5 |
62.5 µg/mL |
- |
3 h |
20 h |
12 |
5 |
125 µg/mL |
- |
3 h |
20 h |
11 |
6 |
Pos. Control |
- |
3 h |
20 h |
41** |
31** |
Negative (Solvent) control |
+ |
3 h |
20 h |
8 |
4 |
test item |
|||||
31.3 µg/mL |
+ |
3 h |
20 h |
10 |
5 |
62.5 µg/mL |
+ |
3 h |
20 h |
12 |
5 |
125 µg/mL |
+ |
3 h |
20 h |
14 |
6 |
180 µg/mL |
+ |
3 h |
20 h |
10 |
5 |
Pos. Control (Cyclophosphamide) |
+ |
3 h |
20 h |
45** |
41** |
Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)
Positive control (+S9): Cyclophosphamide (5.0g/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|||
incl. gaps |
excl. gaps |
||||||
|
Negative (Solvent) control |
- |
20 h |
20 h |
7 |
3 |
|
|
test item |
|
|||||
|
7.8 µg/mL |
- |
20 h |
20 h |
8 |
3 |
|
|
15.6 µg/mL |
- |
20 h |
20 h |
8 |
4 |
|
|
31.3 µg/mL |
- |
20 h |
20 h |
8 |
4 |
|
|
Pos. Control |
- |
20 h |
20 h |
47** |
38** |
|
|
Negative (Solvent) control |
- |
20 h |
28 h |
7 |
3 |
|
|
test item |
|
|||||
|
7.8 µg/mL |
- |
20 h |
28 h |
8 |
3 |
|
|
15.6 µg/mL |
- |
20 h |
20 h |
7 |
3 |
|
|
31.3 µg/mL |
- |
20 h |
28 h |
7 |
4 |
|
|
Pos. Control |
- |
20 h |
28 h |
48** |
37** |
|
Positive control (-S9): Ethyl methanesulphonate (0.4L/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
TABLE 9 continued
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
|
incl. gaps |
excl. gaps |
|
||||
Negative (Solvent) control |
+ |
3 h |
28 h |
7 |
4 |
|
test item |
||||||
31.3 µg/mL |
+ |
3 h |
28 h |
8 |
3 |
|
62.5 µg/mL |
+ |
3 h |
28 h |
10 |
5 |
|
125 µg/mL |
+ |
3 h |
28 h |
10 |
3 |
|
180 µg/mL |
+ |
3 h |
28 h |
11 |
5 |
|
Pos. Control (Cyclophosphamide) |
+ |
3 h |
28 h |
48** |
39** |
|
Cyclophosphamide: 5.0g/mL
**= p < 0.01 to the concurrent negative control and to the historical control
APPENDIX IV
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT A
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
- |
3/20 h |
0.0 |
0.0 |
test item |
||||
31.3 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
62.5 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
125 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
Pos. Control |
- |
3/20 h |
0.0 |
0.0 |
Negative (Solvent) control |
+ |
3/20 h |
0.0 |
0.0 |
test item |
||||
31.3 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
62.5 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
180 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
Pos. Control (Cyclophosphamide) |
+ |
3/20 h |
0.0 |
0.0 |
Ethyl methanesulphonate: 1.0mL/mL
Cyclophosphamide: 5.0g/mL
The number of polyploid and
endoreduplicated cells was determined in
300 cells of each test group.
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT B
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
- |
20/20 h |
0.0 |
0.0 |
test item |
||||
7.8 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
15.6 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
Pos. Control |
- |
20/20 h |
0.0 |
0.0 |
Negative (Solvent) control |
- |
20/28 h |
0.0 |
0.0 |
test item |
||||
7.8 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
15.6 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
Pos. Control |
- |
20/28 h |
0.0 |
0.0 |
Positive control (-S9):Ethyl methanesulphonate(0.4L/mL)
The number of polyploid and
endoreduplicated cells was determined in
300 cells of each test group.
TABLE 11 Continued
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT B
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
+ |
3/28 h |
0.0 |
0.0 |
test item |
||||
31.3 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
62.5 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
180 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
Pos. Control |
+ |
3/28 h |
0.0 |
0.0 |
Cyclophosphamide: 5.0g/mL
The number of polyploid and
endoreduplicated cells was determined in
300 cells of each test group.
Applicant's summary and conclusion
- Conclusions:
- The test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
- Executive summary:
The test item suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473.
Experiment A with 3/20 h treatment/sampling time
without: 15.6,1 31.3, 62.5, 125 and 1802g/mL test item
with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the test item is considered as being non-clastogenic in this system.
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