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EC number: 230-989-6 | CAS number: 7394-38-9
- 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:
- 04 August - 14 September, 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:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: cytogenetic test, which detects structural chromosome aberrations in somatic and/or germ cells
Test material
- Reference substance name:
- p-nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
- EC Number:
- 230-989-6
- EC Name:
- p-nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
- Cas Number:
- 7394-38-9
- Molecular formula:
- C7 H5 N O4 . C6 H15 N O3
- IUPAC Name:
- p-nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
- Test material form:
- solid: particulate/powder
- Details on test material:
- Lot no. 2601LP2609
Storage: At room temperature, protected from light
Constituent 1
Method
- Target gene:
- Chromatid and chromosome type aberrations (gaps, deletions and exchanges), 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).
- Cytokinesis block (if used):
- Colchicine (0.2 µg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver microsome preparations (S9 mix)
- Test concentrations with justification for top dose:
- Cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:
Experiment A with 3/20 h treatment/sampling time
without: 250, 500, 1000 and 2000 *g/mL test item
with S9 mix: 500, 1000 and 2000 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 *g/mL test item - Vehicle / solvent:
- DME (Dulbecco’s Modified Eagle’s) medium
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- 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. 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. Checking for mycoplasma infections was carried out. 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 an incubator with a humidified atmosphere, set at 5 % CO2. The V79 cells for this study was 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%.
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 33.7 and 33.8 mg/mL.
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and ß-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
**= ß-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 is adequate. - 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, a test item is considered to be clearly positive if:
– 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, a test item is considered clearly negative because:
– 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 - 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.
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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Solubility and Dose Selection
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was dissolved in DME (Dulbecco’s Modified Eagle’s). A clear solution was obtained up to a concentration of 50 mg/mL. There was no precipitation in the medium at any concentration tested. Concentration selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays (Experiment A and B), both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by cell counting and results noted. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) are presented in Table 2A - 2C. These results were used to select concentrations of test item for the Chromosome Aberration Assays.
Based on the results of the cytotoxicity assay the following concentrations were selected for the Chromosome Aberration Assay.
All concentrations were run in duplicates (incl. negative and positive controls) and at least 300 (150-150) well-spread metaphases were assessed:
Experiment A with 3/20 h treatment/sampling time
without: 250, 500, 1000 and 2000 *g/mL test item
with S9 mix: 500, 1000 and 2000 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 *g/mL test item
Chromosome Aberration Assay
In Experiment A, P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, neither in the absence nor in the presence of metabolic activation.There were no statistically significant differences between test item treatment and control groups and no dose-response relationship was noted.
In Experiment B, P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent solvent and historical controls.
There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).
A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations without gaps compared to the concurrent solvent and historical controls.
In Experiment A and in Experiment B no statistically significant differences between test item treatment and controls (solvent and historical) groups and no dose-response relationships were noted.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of P-Nitrobenzoic acid,
compound with 2,2’,2”-nitrilotriethanol (1:1).
pH and osmolality values of control and test item treatment solutions were measured. In Experiments A and B no significant differences between test item
treatment and control groups were observed.
In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was less than 5 %, confirming the suitability of the
cell line used.
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 (Tables 3, 4, 5, 6 and 7). Thus, the study is considered valid.
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 |
– |
1950000 |
2050000 |
1968750 |
- |
- |
- |
- |
B |
– |
1950000 |
1950000 |
|||||
- |
C |
– |
2000000 |
2000000 |
|||||
- |
D |
– |
1900000 |
1950000 |
|||||
Solvent control (DME) |
- |
A |
– |
6800000 |
7000000 |
7012500 |
5043750 |
100,00 |
0,00 |
- |
B |
– |
7100000 |
7150000 |
|||||
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
250 |
A |
– |
6900000 |
6900000 |
6900000 |
4931250 |
97,77 |
2,23 |
500 |
A |
– |
5900000 |
6100000 |
6000000 |
4031250 |
79,93 |
20,07 |
|
1000 |
A |
– |
5900000 |
5600000 |
5750000 |
3781250 |
74,97 |
25,03 |
|
2000 |
A |
– |
4100000 |
4250000 |
4175000 |
2206250 |
43,74 |
56,26 |
|
EMS 1µL/mL |
A |
– |
4300000 |
4600000 |
4450000 |
2481250 |
49,19 |
50,81 |
|
Solvent control (DME) medium |
- |
A |
+ |
5900000 |
6250000 |
6162500 |
4193750 |
100,00 |
0,00 |
- |
B |
+ |
6100000 |
6400000 |
|||||
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
250 |
A |
+ |
6150000 |
6150000 |
6150000 |
4181250 |
99,70 |
0,30 |
500 |
A |
+ |
6000000 |
5950000 |
5975000 |
4006250 |
95,53 |
4,47 |
|
1000 |
A |
+ |
5800000 |
5750000 |
5775000 |
3806250 |
90,76 |
9,24 |
|
2000 |
A |
|
5300000 |
5600000 |
5450000 |
3481250 |
83,01 |
16,99 |
|
Cycl. 5µg/mL |
A |
+ |
4100000 |
4000000 |
4050000 |
2081250 |
49,63 |
50,37 |
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 |
– |
1950000 |
2050000 |
1968750 |
- |
- |
- |
- |
B |
– |
1950000 |
1950000 |
|||||
- |
C |
– |
2000000 |
2000000 |
|||||
- |
D |
– |
1900000 |
1950000 |
|||||
Solvent control (DME) |
- |
A |
– |
6500000 |
6200000 |
6362500 |
4393750 |
100,00 |
0,00 |
- |
B |
– |
6300000 |
6450000 |
|||||
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
62.5 |
A |
– |
6400000 |
6200000 |
6300000 |
4331250 |
98,58 |
1,42 |
125 |
A |
– |
6100000 |
6000000 |
6050000 |
4081250 |
92,89 |
7,11 |
|
250 |
A |
– |
5100000 |
4950000 |
5025000 |
3056250 |
69,56 |
30,44 |
|
500 |
A |
– |
4500000 |
4850000 |
4675000 |
2706250 |
61,59 |
38,41 |
|
1000 |
A |
– |
4100000 |
4100000 |
4100000 |
2131250 |
48,51 |
51,49 |
|
2000 |
A |
– |
3200000 |
3500000 |
3350000 |
1381250 |
31,44 |
68,56 |
|
EMS 1µL/mL |
A |
– |
4200000 |
4000000 |
4100000 |
2131250 |
48,51 |
51,49 |
RICC=Relative Increase in Cell Counts
Cytotoxicity= 100-RICC
DME: (Dulbecco’s Modified Eagle’s)medium
EMS: Ethyl methanesulfonate (EMS)
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 |
– |
1950000 |
2050000 |
1968750 |
- |
- |
- |
- |
B |
– |
1950000 |
1950000 |
|||||
- |
C |
– |
2000000 |
2000000 |
|||||
- |
D |
– |
1900000 |
1950000 |
|||||
Solvent control (DME) |
- |
A |
– |
7850000 |
7700000 |
7862500 |
5893750 |
100,00 |
0,00 |
- |
B |
– |
8000000 |
7900000 |
|||||
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
62.5 |
A |
– |
7850000 |
7700000 |
7775000 |
5806250 |
98,52 |
1,48 |
125 |
A |
– |
7500000 |
7450000 |
7475000 |
5506250 |
93,43 |
6,57 |
|
250 |
A |
– |
6100000 |
6000000 |
6050000 |
4081250 |
69,25 |
30,75 |
|
500 |
A |
– |
5400000 |
5750000 |
5575000 |
3606250 |
61,19 |
38,81 |
|
1000 |
A |
– |
4850000 |
4900000 |
4875000 |
2906250 |
49,31 |
50,69 |
|
2000 |
A |
– |
3850000 |
3500000 |
3675000 |
1706250 |
28,95 |
71,05 |
|
EMS 1µL/mL |
A |
– |
4800000 |
4700000 |
4750000 |
2781250 |
47,19 |
52,81 |
|
Solvent control (DME) |
- |
A |
+ |
7850000 |
7650000 |
7787500 |
5806250 |
100,00 |
0,00 |
- |
B |
+ |
7950000 |
7700000 |
|||||
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
250 |
A |
+ |
7750000 |
7750000 |
7750000 |
5768750 |
99,35 |
0,65 |
500 |
A |
+ |
7550000 |
7700000 |
7625000 |
5643750 |
97,20 |
2,80 |
|
1000 |
A |
+ |
7150000 |
7100000 |
7125000 |
5143750 |
88,59 |
11,41 |
|
2000 |
A |
+ |
6900000 |
6750000 |
6825000 |
4843750 |
83,42 |
16,58 |
|
Cycl. 5µg/mL |
A |
+ |
5050000 |
4850000 |
4950000 |
2968750 |
51,13 |
48,87 |
RICC=Relative Increase in Cell Counts
Cytotoxicity= 100-RICC
DME: (Dulbecco’s Modified Eagle’s)medium
EMS: Ethyl methanesulfonate (EMS)
Cycl: Cyclophosphamide monohydrate
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 |
6 |
3 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
|||||
250 µg/mL |
- |
3 h |
20 h |
6 |
4 |
500 µg/mL |
- |
3 h |
20 h |
7 |
4 |
1000 µg/mL |
- |
3 h |
20 h |
8 |
3 |
2000 µg/mL |
- |
3 h |
20 h |
8 |
3 |
Pos. Control |
- |
3 h |
20 h |
37** |
29** |
Negative (Solvent) control |
+ |
3 h |
20 h |
6 |
3 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
|||||
500 µg/mL |
+ |
3 h |
20 h |
9 |
4 |
1000 µg/mL |
+ |
3 h |
20 h |
7 |
4 |
2000 µg/mL |
+ |
3 h |
20 h |
8 |
4 |
Pos. Control (Cyclophosphamide) |
+ |
3 h |
20 h |
44** |
38** |
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 |
6 |
3 |
|
|
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
|
|||||
|
125 µg/mL |
- |
20 h |
20 h |
6 |
3 |
|
|
250 µg/mL |
- |
20 h |
20 h |
7 |
3 |
|
|
500 µg/mL |
- |
20 h |
20 h |
7 |
4 |
|
|
1000 µg/mL |
- |
20 h |
20 h |
6 |
3 |
|
|
Pos. Control |
- |
20 h |
20 h |
45** |
35** |
|
|
Negative (Solvent) control |
- |
20 h |
28 h |
6 |
3 |
|
|
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
|
|||||
|
125 µg/mL |
- |
20 h |
28 h |
6 |
3 |
|
|
250 µg/mL |
- |
20 h |
28 h |
7 |
3 |
|
|
500 µg/mL |
- |
20 h |
20 h |
8 |
4 |
|
|
1000 µg/mL |
- |
20 h |
28 h |
7 |
3 |
|
|
Pos. Control |
- |
20 h |
28 h |
43** |
35** |
|
Positive control (-S9): Ethyl methanesulphonate (0.4L/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 |
+ |
3 h |
28 h |
7 |
4 |
|
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
||||||
500 µg/mL |
+ |
3 h |
28 h |
8 |
2 |
|
1000 µg/mL |
+ |
3 h |
28 h |
7 |
4 |
|
2000 µg/mL |
+ |
3 h |
28 h |
9 |
5 |
|
Pos. Control (Cyclophosphamide) |
+ |
3 h |
28 h |
48** |
38** |
|
Cyclophosphamide: 5.0g/mL
** = p < 0.01 to the concurrent negative control and to the historical control
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 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
||||
250 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
500 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
1000 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
2000 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 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
||||
500 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
1000 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
2000 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 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
||||
125 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
250 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
500 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
1000 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 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
||||
125 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
250 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
500 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
1000 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.
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 |
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) |
||||
500 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
1000 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
2000 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.
HISTORICAL CONTROL DATA
3h/20h treatment/sampling time without S9-mix
|
number of aberrant cells/ 150 cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
5.65 |
2.65 |
40.69 |
31.42 |
SD |
0.71 |
0.65 |
3.44 |
3.67 |
Lower confidence interval |
4.11 |
1.24 |
33.19 |
23.42 |
Upper confidence interval |
7.19 |
4.07 |
48.19 |
39.43 |
n |
13 |
13 |
13 |
13 |
n = number of experiments
SD = standard deviation
3h/20h treatment/sampling time with S9-mix
|
number of aberrant cells/150cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
5.85 |
2.77 |
46.19 |
39.54 |
SD |
0.88 |
0.55 |
2.23 |
2.45 |
Lower confidence interval |
3.94 |
1.56 |
41.34 |
34.20 |
Upper confidence interval |
7.76 |
3.98 |
51.05 |
44.88 |
n |
13 |
13 |
13 |
13 |
n = number of experiments
SD = standard deviation
20h/20h treatment/sampling time without S9-mix
|
number of aberrant cells/150cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
5.62 |
2.77 |
45.77 |
38.19 |
SD |
0.96 |
0.62 |
2.39 |
2.03 |
Lower confidence interval |
3.52 |
1.42 |
40.57 |
33.77 |
Upper confidence interval |
7.71 |
4.12 |
50.97 |
42.61 |
n |
13 |
13 |
13 |
13 |
n = number of experiments
SD = standard deviation
20h/28h treatment/sampling time without S9-mix
|
number of aberrant cells/ 150cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
5.54 |
2.73 |
45.31 |
36.85 |
SD |
0.62 |
0.59 |
2.27 |
2.84 |
Lower confidence interval |
4.19 |
1.45 |
40.36 |
30.65 |
Upper confidence interval |
6.89 |
4.01 |
50.25 |
43.04 |
n |
13 |
13 |
13 |
13 |
n = number of experiments
SD = standard deviation
3h/28h treatment/sampling time with S9-mix
|
number of aberrant cells/ 150 cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
5.65 |
2.73 |
45.81 |
38.58 |
SD |
0.52 |
0.52 |
1.99 |
2.97 |
Lower confidence interval |
4.52 |
1.60 |
41.47 |
32.11 |
Upper confidence interval |
6.78 |
3.86 |
50.14 |
45.04 |
n |
13 |
13 |
13 |
13 |
n = number of experiments
SD = standard deviation
Applicant's summary and conclusion
- Conclusions:
- P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), tested up to the maximum cytotoxic cconcentrations without mammalian metabolic activation system and up to the maximum recommended concentration with mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.
Thus, the test item is considered as not clastogenic in this system. - Executive summary:
The test item, P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was tested in a Chromosome Aberration Assay in V79 cells.The test item was dissolved inDME (Dulbecco’s Modified Eagle’s) mediumand the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using rodent S9 mix).In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:
Experiment Awith 3/20 h treatment/sampling time
without: 250, 500, 1000and 2000g/mLtest item
with S9 mix: 500, 1000 and 2000g/mLtest item
Experiment Bwith 20/20 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000g/mLtest item
Experiment Bwith 20/28 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000g/mLtest item
Experiment Bwith 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000g/mLtest item
In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for lower -cytotoxic substances is 2000 µg/mL (based on the updated OECD Guideline 473 (2016)).
Following treatment (exposure) and sampling (expression) time cells were exposed to selection agent Colchicine
(0.2 µg/mL) 2.5 hours prior to harvesting. Following harvesting cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 300 well-spread metaphase cells.
In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum cytotoxic concentrations and recomemmended concentrations.There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.
In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.
In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.The observed chromosome aberration rates were within the ranges of historical control data.
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.
There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.
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.
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), tested up to the maximum cytotoxic cconcentrations without mammalian metabolic activation system and up to the maximum recommended concentration with mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.
Thus, the test item is considered as not clastogenic in this system.
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