Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates. The studies are as mentioned below:

AMES assay;

Ames assay was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of aroclor 1254 induced S9 metabolic activation system in the standard plate incorporation assay. A dose range finding study was performed at dose levels of 5, 50, 500 and 5000 ug/plate. On the basis of the results obtained from the preliminary study, the test chemical was dissolved in DMSO and used at dose level of 15, 50, 150, 500 and 1500 ug/plate for the main study. In two independent assays with and without metabolic activation no increase of the number of revertants was observed. Based on the observations made, test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

 

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. Typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the Preincubation assay. The test chemical was used at a dosage level of 0, 100, 333, 1000, 3333, 6666 or 10000 µg/plate in the preincubation assay of 48 hrs. Test chemical to induce gene mutation in the S. Typhimurium tester strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

In vitro mammalian gene mutation assay;

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. pH and osmolality was not determined in the gene mutation test. Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 4.53 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of 1-phenylethanol and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs. Based on the results of the current study, we conclude that test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

In an in vitro Mammalian cell gene mutation assay, the mutagenic effects and the antimutagenic activity of test chemical was evaluated in a Chinese hamster V79 cell line. Cells were exposed to 100 µM test chemical alone or in combination with methyl methanesulfonate (MMS), N-nitroso-N-methylurea (MNU), ethyl methanesulfonate (EMS) and UV light. According to the results, test chemical (100µM) did not show any mutagenic or toxic effects by itself. Cinnamaldehyde reduced the mutation frequency induced by MMS but not of the other mutagens. Therefore,test chemical is regarded as not mutagenic.

Based on the data summarized, Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates is expected to not induce gene mutation in mammalian cells and hence it is not likely to be mutagenic in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

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:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, and TA97

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:

S-9 from aroclor 1254 induced rat livers

Test concentrations with justification for top dose:

1,dose range finding: 5, 50, 500 and 5000 ug/platemain assay (with independent repeat): 15, 50, 150, 500 and 1500 ug/plate2,0, 100, 333, 1000, 3333, 6666 or 10000 µg/plate

Vehicle / solvent:

1,- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: no data2,- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The chemical was soluble and stable in DMSO

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

N-ethyl-N-nitro-N-nitrosoguanidine

other: aminoanthrocene

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

2-Aminoanthracene (2-AA).;Sodium azide (SA); 9-aminoacridine (9-AAD)

Details on test system and experimental conditions:

1,METHOD OF APPLICATION: in medium; in agar (plate incorporation)- Cell density at seeding (if applicable):ca 1E07/mLDURATION- Preincubation period: NA- Exposure duration: 72 hours at 37 °CNUMBER OF REPLICATIONS: 3/concentration (2 independent assays)DETERMINATION OF CYTOTOXICITY- Method:reduced bacterial back ground lawn and precipitate.2,Details on test system and conditionsMETHOD OF APPLICATION: preincubationDURATION- Preincubation period: 20 mins- Exposure duration: 48 hrs- Expression time (cells in growth medium): 48 hrs- Selection time (if incubation with a selection agent): No data available

Rationale for test conditions:

No data

Evaluation criteria:

1,A test item is considered as mutagenic if:- a significant and dose-related increase in the number of revertants occurs with sufficient reproducibility- the number of revertant colonies is at least twice as high 2,An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of "+W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. The chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. It chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.

Statistics:

NA

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, and TA97

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- Definition of acceptable cells for analysis: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: dose range finding study was performed at dose levels of 5, 50, 500 and 5000 ug/plateCYTOKINESIS BLOCK (if used)- Distribution of mono-, bi- and multi-nucleated cells: No dataNUMBER OF CELLS WITH MICRONUCLEI- Number of cells for each treated and control culture: No data- Indication whether binucleate or mononucleate where appropriate: No dataHISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data: No data- Negative (solvent/vehicle) historical control data: No dataADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: No data- Other observations when applicable: No data2, RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates 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 ofReaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates. The studies are as mentioned below:

Ames assay was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of aroclor 1254 induced S9 metabolic activation system in the standard plate incorporation assay. A dose range finding study was performed at dose levels of 5, 50, 500 and 5000 ug/plate. On the basis of the results obtained from the preliminary study, the test chemical was dissolved in DMSO and used at dose level of 15, 50, 150, 500 and 1500 ug/plate for the main study. In two independent assays with and without metabolic activation no increase of the number of revertants was observed. Based on the observations made, test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. Typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the Preincubation assay. The test chemical was used at a dosage level of 0, 100, 333, 1000, 3333, 6666 or 10000 µg/plate in the preincubation assay of 48 hrs. Test chemical to induce gene mutation in the S. Typhimurium tester strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

Based on the data summarized, Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates 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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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 for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

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:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot. This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).- Properly maintained: Yes- Periodically checked for Mycoplasma contamination: Not applicable- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's MEM supplemented with 5% fetal calf serum(FCS), 100 U/ml penicillin G and 100 µg/mlstreptomycin sulfate- Properly maintained: Yes- Periodically checked for Mycoplasma contamination: No data available- 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):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

1,0, 0.5, 1, 2.5 or 5 mM2,100 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)- Justification for choice of solvent/vehicle: 1-phenylethanol was easily dissolved in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: N-nitroso-N-methyl-urea (MNU)

Details on test system and experimental conditions:

1,METHOD OF APPLICATION: In medium with pre-incubationDURATION- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours. - Exposure duration: 3 hours- Expression time (cells in growth medium): 7 days- Selection time (if incubation with a selection agent): 14 days- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)SELECTION AGENT (mutation assays): 6-thioguanine (TG)SPINDLE INHIBITOR (cytogenetic assays): Not applicableSTAIN (for cytogenetic assays): Crystal violetNUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluatedDETERMINATION OF CYTOTOXICITY- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding. OTHER EXAMINATIONS: Not applicable - Determination of polyploidy:- Determination of endoreplication:- Other:OTHER:2,METHOD OF APPLICATION: In mediumDURATION- Preincubation period: No data available- Exposure duration: 2-4 hrs- Expression time (cells in growth medium): 7 days- Selection time (if incubation with a selection agent): 10 days- Fixation time (start of exposure up to fixation or harvest of cells): 7 daysSELECTION AGENT (mutation assays): 6-ThioguanineSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: 3-4NUMBER OF CELLS EVALUATED: 5 × 105 cells/100-mm dish; For cell viability: 100 cells/60-mm dish DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Cell viability OTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

Not specified

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

Positive controls validity:

not valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic effct were observed

Conclusions:

The test chemical Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates does not induce gene mutation in mammlian cell 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 Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates. The studies are as mentioned below:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. pH and osmolality was not determined in the gene mutation test. Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 4.53 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of 1-phenylethanol and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs. Based on the results of the current study, we conclude that test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

In an in vitro Mammalian cell gene mutation assay, the mutagenic effects and the antimutagenic activity of test chemical was evaluated in a Chinese hamster V79 cell line. Cells were exposed to 100 µM test chemical alone or in combination with methyl methanesulfonate (MMS), N-nitroso-N-methylurea (MNU), ethyl methanesulfonate (EMS) and UV light. According to the results, test chemical (100µM) did not show any mutagenic or toxic effects by itself. Cinnamaldehyde reduced the mutation frequency induced by MMS but not of the other mutagens. Therefore,test chemical is regarded as not mutagenic.

Based on the data summarized, Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates is expected to not induce gene mutation in mammalian cells 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

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates. The studies are as mentioned below:

AMES assay;

Ames assay was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of aroclor 1254 induced S9 metabolic activation system in the standard plate incorporation assay. A dose range finding study was performed at dose levels of 5, 50, 500 and 5000 ug/plate. On the basis of the results obtained from the preliminary study, the test chemical was dissolved in DMSO and used at dose level of 15, 50, 150, 500 and 1500 ug/plate for the main study. In two independent assays with and without metabolic activation no increase of the number of revertants was observed. Based on the observations made, test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

 

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. Typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the Preincubation assay. The test chemical was used at a dosage level of 0, 100, 333, 1000, 3333, 6666 or 10000 µg/plate in the preincubation assay of 48 hrs. Test chemical to induce gene mutation in the S. Typhimurium tester strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

In vitro mammalian gene mutation assay;

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. pH and osmolality was not determined in the gene mutation test. Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 4.53 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of 1-phenylethanol and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs. Based on the results of the current study, we conclude that test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

In an in vitro Mammalian cell gene mutation assay, the mutagenic effects and the antimutagenic activity of test chemical was evaluated in a Chinese hamster V79 cell line. Cells were exposed to 100 µM test chemical alone or in combination with methyl methanesulfonate (MMS), N-nitroso-N-methylurea (MNU), ethyl methanesulfonate (EMS) and UV light. According to the results, test chemical (100µM) did not show any mutagenic or toxic effects by itself. Cinnamaldehyde reduced the mutation frequency induced by MMS but not of the other mutagens. Therefore,test chemical is regarded as not mutagenic.

Based on the data summarized, Reaction mass of Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphates is expected to not induce gene mutation in mammalian cells 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 Benzenamine, N,N-dimethyl- , molybdate, tungstate & phosphatesdoes not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.