[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES Assay

Test chemical  did not induce gene 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 vitro gene mutation study in mammalian cell

Test chemical was evaluated for its mutagenic potential in mammalian cells by in vitro mammalian cell gene mutation. The test result was considered to be negative both in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

Justification for type of information:

data from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

Weight of evidence prepared from various publication mention below
Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA1538, TA98 and TA100

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

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/plate
2,0.1 mg/plate or 1 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: No data
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

No data

Details on test system and experimental conditions:

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 data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER 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 data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

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.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

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:

not specified

Species / strain:

S. typhimurium, other: TA1538, TA98 and TA100

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:

not specified

Additional information on results:

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 data

RANGE-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 data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Test chemical did not induce gene 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.

Executive summary:

Data for test chemicals was reviewed to determine the mutagenic nature of [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7). The studies are as mentioned below:

AMES Assay

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of test substance. 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 S9 metabolic activation system. Concurrent solvent and positive controls were used in the study. Test chemical did not induce gene mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation study was performed to evaluate the mutagenic nature of test substance using Salmonella typhimurium strain TA1538 and TA100. Bacteria were grown overnight in Oxoid nutrient broth, then refrigerated at 4-5OC for a few hours before use. 0.1 ml of bacterial culture was added to 2 ml of 45°C molten top agar containing 0.01 mg histidine HCI and 0.012 mg biotin/ml, followed by the test sample in ≤0.2 ml DMSO. Finally, 0.5 ml of sodium phosphate buffer, pH 7.4 (no activation), or 0.5 ml of Aroclor-induced rat S9 mixture was added, and the mixture was poured on minimal glucose agar plates. Histidine revertant colonies were counted on a Biotran II automated colony counter after 2-day incubation at 37°C. A sample was judged mutagenic if it produced greater than twice the spontaneous background colonies at more than one dose or at the highest dose tested. In the above mentioned study, test substance failed to induce gene mutation in the Salmonella typhimurium strains TA1538 and TA100 with and without metabolic activation. Hence, test substance, is not likely to be a gene mutant 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

Justification for type of information:

data from handbook or collection of data.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

Weight of evidence prepared from various publication mention below
1,To evaluate the mutagenic potential of test substance in CHINESE HAMSTER V-79 by in vitro mammalian cell gene mutation assay.
2,To evaluate the mutagenic potential of test substance in Chinese hamster ovary by In vitro mammalian cell gene mutation assay

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian cell gene mutation assay

Target gene:

HGPRT locus (6-thioguanine resistance)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

An Kanechlor 400 induced rat liver S9 fraction

Test concentrations with justification for top dose:

1,0.3-33µg/mL
2,4.64-215 µg/mL

Vehicle / solvent:

Yes, not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

1,METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 5 hour
NUMBER OF CELLS EVALUATED: 3 x 106
SELECTION AGENT (mutation assays): 6-TG, 60 µM
DETERMINATION OF CYTOTOXICITY
- Method: other: not specified

2,METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4hour


NUMBER OF CELLS EVALUATED: 5 x 105

SELECTION AGENT (mutation assays): 6-TG, 10 µM


DETERMINATION OF CYTOTOXICITY
- Method: other: not specified

Evaluation criteria:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 5 hour


NUMBER OF CELLS EVALUATED: 3 x 106

SELECTION AGENT (mutation assays): 6-TG, 60 µM


DETERMINATION OF CYTOTOXICITY
- Method: other: not specified


The mammalian cells were observed for mutagenic effects.

Statistics:

Yes SD± Mean was observed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No toxic potential was observed

Conclusions:

Test chemical was evaluated for its mutagenic potential in mammalian cells by in vitro mammalian cell gene mutation. The test result was considered to be negative both in the presence and absence of metabolic activation.

Executive summary:

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian cell gene mutation was performed in Chinese hamster cells. The test chemical was exposed to the Chinese hamster cell sin the presence and absence of metabolic activation. Different concentration of test material used on cells were in µg/mL. No mutagenic frequency were observed in the mammalian cells in the presence and absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic in Chinese hamster cells by in vitro mammalian cell gene mutation. Hence the substance cannot be classified as 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 test chemicals was reviewed to determine the mutagenic nature of [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7). The studies are as mentioned below:

AMES Assay

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of test substance. 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 S9 metabolic activation system. Concurrent solvent and positive controls were used in the study. Test chemical did not induce gene mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation study was performed to evaluate the mutagenic nature of test substance using Salmonella typhimurium strain TA1538 and TA100. Bacteria were grown overnight in Oxoid nutrient broth, then refrigerated at 4-5OC for a few hours before use. 0.1 ml of bacterial culture was added to 2 ml of 45°C molten top agar containing 0.01 mg histidine HCI and 0.012 mg biotin/ml, followed by the test sample in ≤0.2 ml DMSO. Finally, 0.5 ml of sodium phosphate buffer, pH 7.4 (no activation), or 0.5 ml of Aroclor-induced rat S9 mixture was added, and the mixture was poured on minimal glucose agar plates. Histidine revertant colonies were counted on a Biotran II automated colony counter after 2-day incubation at 37°C. A sample was judged mutagenic if it produced greater than twice the spontaneous background colonies at more than one dose or at the highest dose tested. In the above mentioned study, test substance failed to induce gene mutation in the Salmonella typhimurium strains TA1538 and TA100 with and without metabolic activation. Hence, test substance, is not likely to be a gene mutant in vitro.

In vitro gene mutation study in mammalian cell

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian cell gene mutation was performed in Chinese hamster V-79 cells. The test chemical was exposed to the Chinese hamster V-79 cell sin the presence and absence of metabolic activation. The concentration of test material used on cells were 0.3-33µg/mL. No mutagenic frequency were observed in the mammalian cells in the presence and absence of metabolic activation. Therefore test chemical was

considered to be non-mutagenic in Chinese hamster V-79 calls by in vitro mammalian cell gene mutation. Hence the substance cannot be classified as mutagenic in vitro.

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian cell gene mutation was performed in Chinese hamster ovary cells. The test chemical was exposed to the Chinese hamster V-79 cells in the presence and absence of metabolic activation. The concentration of test material used on cells were 4.64-215 µg/mL. No mutagenic frequency were observed in the mammalian cells in the presence and absence of metabolic activation. Therefore test chemical was

considered to be non-mutagenic in Chinese hamster ovary cells by in vitro mammalian cell gene mutation. Hence the substance cannot be classified as mutagenic in vitro.

Based on the data summarized, [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7)is expected to not induce gene mutation In vitro. 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 [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7)  does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.