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EC number: 209-218-2 | CAS number: 561-41-1
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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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study contains experimental data of the registered substance.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol
- EC Number:
- 209-218-2
- EC Name:
- 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol
- Cas Number:
- 561-41-1
- Molecular formula:
- C24-H29-N3-O
- IUPAC Name:
- bis[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methanol
- Test material form:
- solid
- Details on test material:
- CAS No. : 561-41-1
Ec No. 209-218-2
Appearance : Violet powder
Batch Number /Lot Number : KCP/FS/199/21
Purity / AI Content : 98.81%
Manufactured by : K. Patel Chemopharma Pvt. Ltd.
Manufacturing date : 26.05.2021
Expiry Date : 25.05.2022
Storage condition : Room Temperature (20 to 30oC)
Safety precautions : Standard safety precautions (gloves, mask, apron, head cap and goggles).
Constituent 1
- Specific details on test material used for the study:
- CAS No. : 561-41-1
Ec No. 209-218-2
Appearance : Violet powder
Batch Number /Lot Number : KCP/FS/199/21
Purity / AI Content : 98.81%
Manufactured by : K. Patel Chemopharma Pvt. Ltd.
Manufacturing date : 26.05.2021
Expiry Date : 25.05.2022
Storage condition : Room Temperature (20 to 30oC)
Safety precautions : Standard safety precautions (gloves, mask, apron, head cap and goggles).
Method
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO cells were cultured in complete RPMI 1640 medium (10 % fetal bovine serum) and maintained at 37 ±2 °C, 5% CO2 in the CO2 incubator. Cells were counted, and the volume was then adjusted with fresh media to obtain a cell density of 2 x 105 cells/25 cm2 and incubated until attainment of 1 x 106 cells for cytotoxicity measurement and chromosomal aberrations. Cells free from mycoplasma were used in the study.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Homogenate
A combination of phenobarbitone and β-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate) prepared in-house was used for the assay. - Test concentrations with justification for top dose:
- The test concentrations were selected based on the solubility and precipitation checks and a preliminary cytotoxicity test.
The cytotoxicity of the Test Item was evaluated both in the presence and absence of a metabolic activation system. Five different concentrations of the Test Item 0.125, 0.25, 0.5, 1 and 2 mg/ml along with negative and vehicle controls were used by using a single culture.
In the presence and absence of metabolic activation, cells were treated with the Test Item, negative, and vehicle controls with and without metabolic activation for 4 hours (short term treatment). At the end of the incubation, cell counts were determined to calculate the Relative Increase in Cell Counts (RICC).
The cytotoxicity was evaluated based on the reduction in the RICC. A Test Item concentration causing approximately 45±5% reduction in RICC to that of the concurrent vehicle control (i.e. 55±5% cytotoxicity) was selected as the highest test concentration for the chromosome aberration assay. Two lower concentrations, using spacing factor 2, were included in the assay. - Vehicle / solvent:
- dimethyl sulfoxide
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments - 3 (Priliminary cytology, Phase I, Phase II and Phase III)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 106
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk - Test substance added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Cells were counted, and the volume was then adjusted with fresh media to obtain a cell density of 2 x 105 cells/25 cm2 and incubated until attainment of 1 x 106 cells.
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): The cultures were harvested 24 hours after the beginning of treatment.
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.- Two hours prior to harvesting, a volume of 50 µl from 0.1 mg/ml of colchicine stock was added to culture flask to achieve a final concentration of 1 µg/ml and incubated at 37 ±2 °C, 5 % CO2 in a CO2 incubator.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. - No
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Slides were stained with freshly prepared 5 % Giemsa stain for 5 minutes and rinsed in distilled water for 2 minutes. After drying, slides were mounted using DPX mountant.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): As slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, cells that contain the number of centromeres equal to the number 2n ± 2 were scored. In CHO cells, the number of centromeres equal to the modal number is 22 ± 2.
- Determination of polyploidy:
- Determination of endoreplication:
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: relative increase in cell count (RICC)
- Any supplementary information relevant to cytotoxicity:
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER: - Evaluation criteria:
- 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 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 of the distribution of the laboratory historical negative control database
Test Item is considered clearly negative if, in all experimental conditions examined:
None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
There is no concentration-related increase when evaluated with an appropriate trend test.
The results are inside the distribution of the laboratory historical negative control database. - Statistics:
- Statistical analysis was performed to assess a possible dose-dependent increase of aberrant cell frequencies using Fisher’s Exact Test (NCSS statistics software). The percentage of aberrant cells from the Test Item treated group was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- In the initial preliminary cytotoxicity assay (Preliminary cytotoxicity assay-I), excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.125 mg/ml, both in the presence and absence of metabolic activation.
Since, an excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed during the initial preliminary cytotoxicity assay in all the tested concentrations, an additional preliminary cytotoxicity assay (Preliminary cytotoxicity assay-II) was performed with the Test Item at the following concentrations, 0.0625, 0.0125, 0.025, 0.05 and 0.1 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with vehicle and negative controls.
In this cytotoxicity assay (Preliminary cytotoxicity assay-II), excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. RICC decreased by 55±5% compared to vehicle control at 0.00625 mg/ml both in the presence and absence of metabolic activation. RICC were 46.86% (cytotoxicity: 52.92%) and 42,69% (cytotoxicity: 57.31) in the absence and presence of S9 metabolic activation system, respectively. - Remarks on result:
- other: No mutagenic potential was observed
Any other information on results incl. tables
Appendix1: RelativeIncrease in Cell Counts – Preliminary Cytotoxicity Assay
Dose Level |
Conc. (mg/ml) |
Absence of Metabolic activation |
Presence of Metabolic activation |
||||||
Cell count |
RICC |
% Cytotoxicity |
Cell count |
RICC |
% Cytotoxicity |
||||
Starting |
Final |
Starting |
Final |
||||||
NC |
Distilled water |
1000000 |
3580000 |
100.00 |
0.00 |
1000000 |
3584000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3560000 |
99.22 |
0.78 |
1000000 |
3470000 |
95.59 |
4.41 |
T1 |
0.125 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
T2 |
0.25 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
T3 |
0.5 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
T4 |
1 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
T5 |
2 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
Key: NC = Negative Control, VC = Vehicle Control, Conc. = Concentration, mg = milligram, ml = milliliter, RICC = Relative Increase in Cell Counts, % = percentage.
Appendix3: Relative Increase in Cell Counts- Main Study
Dose Level |
Conc.
|
Phase I -Absence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
NC |
Distilled water |
1000000 |
3586000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3530000 |
97.83 |
2.17 |
T1 |
0.0015625 mg/ml |
1000000 |
2742000 |
68.85 |
31.15 |
T2 |
0.003125 mg/ml |
1000000 |
2342000 |
53.04 |
46.96 |
T3 |
0.00625 mg/ml |
1000000 |
2160000 |
45.85 |
54.15 |
PC |
20 µg/ml |
1000000 |
2842000 |
72.81 |
27.19 |
Dose Level |
Conc. |
Phase II -Presence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
NC |
Distilled water |
1000000 |
3550000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3460000 |
96.47 |
3.53 |
T1 |
0.0015625 mg/ml |
1000000 |
2644000 |
66.83 |
33.17 |
T2 |
0.003125 mg/ml |
1000000 |
2386000 |
56.34 |
43.66 |
T3 |
0.00625 mg/ml |
1000000 |
2018000 |
41.38 |
58.62 |
PC |
30 µg/ml |
1000000 |
2642000 |
66.75 |
33.25 |
Dose Level |
Conc. |
Phase III -Absence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
NC |
Distilled water |
1000000 |
3582000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3460000 |
95.27 |
4.73 |
T1 |
0.0015625 mg/ml |
1000000 |
2582000 |
64.31 |
35.69 |
T2 |
0.003125 mg/ml |
1000000 |
2282000 |
52.11 |
47.89 |
T3 |
0.00625 mg/ml |
1000000 |
2024000 |
41.63 |
58.37 |
PC |
20 µg/ml |
1000000 |
2682000 |
68.37 |
31.63 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control, Conc. = Concentration, mg = milligram, ml = milliliter, µg = microgram, RICC = Relative Increase in Cell Counts, % = percentage.
Appendix7: Summary Dataon Chromosome Aberrations - Phase I
Dose Level |
Concentration
|
Absence of metabolic activation |
|
Total No. of Aberrant cells without gap |
Percent aberrant cells |
||
NC |
Distilled water |
0 |
0.00 |
VC |
DMSO |
0 |
0.00 |
T1 |
0.0015625 mg/ml |
2 |
0.67 |
T2 |
0.003125 mg/ml |
1 |
0.33 |
T3 |
0.00625 mg/ml |
1 |
0.33 |
PC |
20 µg/ml* |
24 |
8.00 |
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05).
Appendix8:Summary Data on Chromosome Aberrations - Phase II
Dose Level |
Concentration
|
Presence of metabolic activation |
|
Total No. of Aberrant cells without gap |
Percent aberrant cells |
||
NC |
Distilled water |
0 |
0.00 |
VC |
DMSO |
0 |
0.00 |
T1 |
0.0015625 mg/ml |
2 |
0.67 |
T2 |
0.003125 mg/ml |
0 |
0.00 |
T3 |
0.00625 mg/ml |
2 |
0.67 |
PC |
30 µg/ml* |
21 |
7.00 |
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05).
Appendix9:
SummaryData
on Chromosome Aberrations - Phase III
Dose Level |
Concentration |
Absence of metabolic activation |
|
Total No. of Aberrant cells without gap |
Percent aberrant cells |
||
NC |
Distilled water |
0 |
0.00 |
VC |
DMSO |
0 |
0.00 |
T1 |
0.0015625 mg/ml |
1 |
0.33 |
T2 |
0.003125 mg/ml |
1 |
0.33 |
T3 |
0.00625 mg/ml |
2 |
0.67 |
PC |
20 µg/ml* |
27 |
9.00 |
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05).
Applicant's summary and conclusion
- Conclusions:
- Based on the results of this study, it is concluded that 4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1] did not induce chromosomal aberration up to the highest concentration of 0.00625 mg/ml of culture medium, either in the presence or absence of a metabolic activation system in CHO cells under the experimental conditions described.
- Executive summary:
Thisin vitro experiment was performed, to evaluatethe ability of4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1]to cause structural chromosomal aberrations in cultured CHO cells in the presence and absence of an exogenous metabolic activation system (S9).The study was performed as per OECD Test Guidelines No.473 (Adopted: 29 July 2016).
The Test Item, i.e.4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1],was found to be soluble at 200 mg/ml concentration in dimethyl sulfoxide. Slight precipitation was observed at the concentration of 2 mg/ml. The pH values of the test concentrations in media are mentioned below.
Test Item
Concentration
pH
0 hour
Incubation
4 hours
Incubation
2 mg/ml
7.4
7.4
Based on the solubility and precipitation tests, an initial preliminary cytotoxicity testing was performed with the Test Item at the following concentrations, 0.125, 0.25, 0.5, 1 and 2 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with vehicle and negative controls.
In the initial preliminary cytotoxicity assay (Preliminary cytotoxicity assay-I), excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at≥0.125mg/ml, both in the presence and absence of metabolic activation.
Since excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed during the initial preliminary cytotoxicity assay in all tested concentrations, an additional preliminary cytotoxicity assay (Preliminary cytotoxicity assay-II) was performed with the Test Item at the following concentrations, 0.0625, 0.0125, 0.025, 0.05 and 0.1 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with vehicle and negative controls.
In this second cytotoxicity assay(Preliminary cytotoxicity assay-II), excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at≥0.0125mg/ml, both in the presence and absence of metabolic activation.RICC decreased by 55±5% compared to vehicle control at 0.00625 mg/ml both in the presence and absence of metabolic activation. RICC were 46.86% (cytotoxicity: 53.14% and 42,69% (cytotoxicity: 57.31) in the absence and presence of S9 metabolic activation system, respectively.
Based on the cytotoxicity assay results, Phase I (short term exposure in the absence of metabolic activation), Phase II (short term exposure in the presence of metabolic activation) and Phase III (continuous exposure in the absence of metabolic activation) were conducted with the Test Item at the concentrations of 0.0015625, 0.003125 and 0.00625 mg/ml, along with vehicle, negative and concurrent positive controls.
In Phase I, cultures were exposed to Test Item, vehicle, negative and positive control for 4 hours (short term exposure) in the absence of metabolic activation. Average RICC values were 97.83 % (vehicle control), 68.85 % (at 0.0015625 mg/ml), 53.04 % (at 0.003125 mg/ml) and 45.85 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), 0.003125 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.00625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %).
In Phase II, cultures were exposed to Test Item, vehicle, negative and positive control for 4 hours (short term exposure) in the presence of metabolic activation (1 % v/v S9 mix). Average RICC values were 96.47 % (vehicle control), 66.83 % (at 0.0015625 mg/ml), 56.34 % (at 0.003125 mg/ml) and 41.38 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), 0.003125 mg/ml (the mean % aberrant cells: 0.00%, p=1.0000), 0.00625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %).
In Phase III, cultures were exposed to Test Item, vehicle, negative and positive control for 24 hours (continuous exposure) in the absence of metabolic activation. Average RICC values were 95.27 % (vehicle control), 64.31 % (at 0.0015625 mg/ml), 52.11 % (at 0.003125 mg/ml) and 41.63 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.003125 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 0.00625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %).
In all phases of the study, no significant reduction in RICC (cytotoxicity) and no increase in percent aberrant cells were observed in vehicle control (DMSO) either in the presence or absence of metabolic activationwhen compared to the negative control (distilled water).
Based on the results of this study, it is concluded that4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1]does not induce chromosomal aberration up to 0.00625 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system in CHO cells under the experimental conditions described.
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