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Genetic toxicity in vitro

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Genetic toxicity in vitro:

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of Methylium, tris[4-(diethylamino)phenyl]-& acetate. The studies are as mentioned below:

AMES assay;

 

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical. The study was performed at dose levels of 0.3-100 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system. Concurrent solvent and positive controls were used in the study. The test chemical did not induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation and standard plate incorporation assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without S9 metabolic activation system. The liquid preincubation assays were timed for 30 min at 37°C in a Dri-block. The test chemical was dissolved in DMSO and used upto a maximum nontoxic dose of 10 µg/plate. Concurrent solvent and positive controls were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In Vitro Mammalian gene mutation assay;

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of structurally and functionally similar test chemical.The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 0.001 -6 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence andabsence of S9 activation system and hence is not likely to be gene mutant in vitro.

 

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from250-3000µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, Reaction mass of Methylium, tris[4-(diethylamino)phenyl]-& acetateis expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
experimental data of read across substances
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar read across chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
WoE derived based on the experimental data from structurally and functionally similar read across chemicals
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Specific details on test material used for the study:
- Name of the test material (IUPAC name): Tris[4-(diethylamino)phenyl]methylium acetate- Common name: Basic Violet 4- Molecular weight: 515.7375- Molecular formula: C33H45N3O2- Substance type: Organic- Physical Appearance: greenish lustrous liquid- Stability: Stable in recommended storage conditions- Storage Conditions: Ambient Temp (23 to 27 °C)
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Remarks:
1/ 2 / 3
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight
Test concentrations with justification for top dose:
1. 0.3- 100 µg/plate2/3. Maximum nontoxic dose tested was 10 µg/plate
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: No data- Justification for choice of solvent/vehicle: No data2/3. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
No specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Remarks:
2/ 3
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data- Exposure duration: 48 hrs- Expression time (cells in growth medium): 48 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: Five doses of test chemical were tested in triplicate on each tester strain without and with metabolic activation.NUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data2. METHOD OF APPLICATION: preincubationDURATION- Preincubation period: 30 mins- Exposure duration: No data- Expression time (cells in growth medium): No data- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays):SPINDLE INHIBITOR (cytogenetic assays):STAIN (for cytogenetic assays):NUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: The revertant colonies were counted by using a hand-held tally.3. METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data- Exposure duration: No data- Expression time (cells in growth medium): No data- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays):SPINDLE INHIBITOR (cytogenetic assays):STAIN (for cytogenetic assays):NUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data
Rationale for test conditions:
1. No data2. No data3. No data
Evaluation criteria:
1. For a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.2/3. A compound was considered mutagenic when the number of revertants above background was at least twice the value of the historical control mean or twice the value of the current control mean, whichever was greater
Statistics:
1. No data2. No data3. No data
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Remarks:
1/ 2 /3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data - Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.COMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data2/3. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate does not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of Methylium, tris[4-(diethylamino)phenyl]-& acetate. The studies are as mentioned below:

 

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical. The study was performed at dose levels of 0.3-100 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system. Concurrent solvent and positive controls were used in the study. The test chemical did not induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation and standard plate incorporation assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without S9 metabolic activation system. The liquid preincubation assays were timed for 30 min at 37°C in a Dri-block. The test chemical was dissolved in DMSO and used upto a maximum nontoxic dose of 10 µg/plate. Concurrent solvent and positive controls were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data summarized, Reaction mass of Methylium, tris[4-(diethylamino)phenyl]-& acetateis expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
WoE derived based on the experimental data from structurally and functionally similar read across chemicals
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of the test material (IUPAC name): Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate- Common name: Basic Violet 4- Molecular weight: 515.7375- Molecular formula: C33H45N3O2- Substance type: Organic- Physical Appearance: greenish lustrous liquid- Stability: Stable in recommended storage conditions- Storage Conditions: Ambient Temp (23 to 27 °C)
Target gene:
No data
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
TK+/- 3.7.C / 2
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.- Properly maintained: No data available - Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats.
Test concentrations with justification for top dose:
1. 0.001-6 µg/mL2,250-3000 µg/mL
Vehicle / solvent:
1.- Vehicle(s)/solvent(s) used: Water- Justification for choice of solvent/vehicle: The test chemical was soluble in water2. No data available
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
methylmethanesulfonate
other: 3-methylcholanthrene and dimethylbenz[a]- anthracene (+S9)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
ethyl methylsulfonate at 4.7×10-6 M (or methyl methanesulfonate at 10-20 µg/ml mL) for the test without metabolic activation. 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.
Details on test system and experimental conditions:
1, METHOD OF APPLICATION: in mediumCells at start: 6000000 cellsDURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium):48 h- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selectionSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200cells/plate for viable count determinations DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated culturesOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available2,Details on test system and conditionsMETHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium):48 h
Rationale for test conditions:
No data
Evaluation criteria:
1 and 2 Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Statistics:
Not specified
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
TK+/- 3.7.C / 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate did not induce chromosome aberrations and gene mutation in the cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate and as these test chemicals are read across of this target chemical .The read across substances share high similarity in functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. The studies are as mentioned below:

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of structurally and functionally similar test chemical.The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 0.001 -6 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence andabsence of S9 activation system and hence is not likely to be gene mutant in vitro.

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from250-3000µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate is expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

AMES assay;

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of Methylium, tris[4-(diethylamino)phenyl]-& acetate. The studies are as mentioned below:

 

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical. The study was performed at dose levels of 0.3-100 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system. Concurrent solvent and positive controls were used in the study. The test chemical did not induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation and standard plate incorporation assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without S9 metabolic activation system. The liquid preincubation assays were timed for 30 min at 37°C in a Dri-block. The test chemical was dissolved in DMSO and used upto a maximum nontoxic dose of 10 µg/plate. Concurrent solvent and positive controls were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In Vitro Mammalian gene mutation assay;

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of structurally and functionally similar test chemical.The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 0.001 -6 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence andabsence of S9 activation system and hence is not likely to be gene mutant in vitro.

 

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from250-3000µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, Reaction mass of Methylium, tris[4-(diethylamino)phenyl]-& acetateis expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria for target substance Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​& acetatedoes not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.