Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant.

In vitro chromosome abbreviation assay

The test chemical  did not induce chromosome aberrations with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

In vitro Mammalian gene mutation assay

In a gene toxicity test,  mammalian cells were exposed to the test chemical  with and without S9-induced metabolic activation. The results showed no evidence of gene toxicity when cells were exposed to test chemical both in the presence and absence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

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
Justification for type of information:
data from handbook or collection of data
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mention below
Principles of method if other than guideline:
Weight of evidence prepared from various publication mention below
1,This study was performed to investigate the potential of the test chemical to induce gene muta¬tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
2,The preincubation assay was performed . The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37"C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel- Bonner medium [Vogel and Bonner, 19561. The histidine-revertant (his') colonies arising on these plates were counted following 2 days incubation at 37°C. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 metabolic activation system
Test concentrations with justification for top dose:
1,0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate
2,0, 3.3, 10.0, 33.0, 100.0, 200.0, 333.0, 667.0, 1000.0, 3333.0, 6666.0, 6667.0, 10000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble 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:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- 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: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
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
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
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:
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 data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.001 – 2.5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 2.5 mg/plate (T8), 0.791 mg/plate (T7) and no reduction in colony count as well as in background lawn in treated concentrations (0.250 (T6) mg/plate – 0.001 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: (0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL 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 data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of 5-methoxy-1,3-dihydro-2H-benzimidazole-2-thione( 37052-78-1). The studies are as mentioned below:

AMES test

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether (CAS no. 93-04-9) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

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 compound using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs.  The test compound was used at a dosage level of 0, 3.3, 10.0, 33.0, 100.0, 200.0, 333.0, 667.0, 1000.0, 3333.0, 6666.0, 6667.0, 10000.0 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study.

 

Test substance did not induce a reproducible, dose-related increase in his+revertants over the corresponding solvent in the S. typhimurium tester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration 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 from handbook or collection of data
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 prepared from various publication mention below
GLP compliance:
not specified
Type of assay:
other: In vitro chromosomal aberration test
Target gene:
No data
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes
Remarks:
1
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: Human peripheral blood lymphocytes
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
1,0.00 (NC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL
2, 0, 0.33, 1.0, 3.3, 10 mM
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was soluble in distilled water

2. No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
1
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
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data available
- Exposure duration: No data available
- Expression time (cells in growth medium): No data available
- 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 available

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

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data available

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

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

OTHER: No data available.

2. No data
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for chromosome aberration
Statistics:
No data
Species / strain:
lymphocytes: Human peripheral blood lymphocytes
Remarks:
1
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
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
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:
not specified
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 data

RANGE-FINDING/SCREENING STUDIES: Test
concentrations were selected based on the results of a preliminary cytotoxicity test

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.
Executive summary:

Data available for the test chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Human peripheral blood lymphocytes with and without S9 metabolic activation system. The test chemical was dissolved in distilled water and used at dose level of0, 0.33, 1.0, 3.3, 10 mM. Concurrent solvent and positive control plates were also included in the study. The test chemical did not induce chromosome aberrations in Human peripheral blood lymphocytes with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

In another study, an in vitro mammalian cell gene Mutation test was carried out according to OECD guideline 476 using Chinese hamster ovary cells to evaluate the genetic potential of the test chemical. In results, The test chemical did not induced any chromosomal aberrations at concentrations up to 150-1000 μg/plate and 50-1000 μg/plate in Chinese hamster ovary cells 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 observations made, the test chemical 1H-benzimidazole (CAS no 51-17-2) did not induce chromosome aberrations with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
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:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mention below
Principles of method if other than guideline:
WoE prepared from various publication
1,The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
2,To evaluate the mutagenic potential of test chemical in mouse lymphoma L5178Y cells by In vitro gene mutation mammalian cell assay.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
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:
mouse lymphoma L5178Y cells
Cytokinesis block (if used):
No data
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, 0.1, 0.25, 0.5 or 1 mM
2,0- 100 µg/mL
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl 2-napthyl ether was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- 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 applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER 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 evaluated.

DETERMINATION 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
Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for total number of colonies
Statistics:
No data
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:
mouse lymphoma L5178Y cells
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
Remarks on result:
other: No mutagenic potential
Conclusions:
In a gene toxicity test, mammalian cells were exposed to the test chemical with and without S9-induced metabolic activation. The results showed no evidence of gene toxicity when cells were exposed to test chemical both in the presence and absence of metabolic activation.
Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of 5-methoxy-1,3-dihydro-2H-benzimidazole-2-thione( 37052-78-1). The studies are as mentioned below:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, test substance was administered to CHO cells for 3 hrs at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 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 asN-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. 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 was seen in one well out of four at the concentration of 0.5 mM and in the absence with 4% S9 liver microsomal fraction, and two very diffuse colonies were detected in one well out of four at the concentration of 0.1 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the three spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 5.35 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 methyl 2-napthyl ether in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs. Based on the results of the current study, we conclude that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs, in the presence or absence of metabolic activation.

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian cell gene mutation was performed .The test material was exposed tomouse lymphomaL5178Y cells. The concentration of test material used in the concentration of 0- 100 µg/mL. No mutagenic frequency were observed in the mammalian cells in presence and absence of metabolic activation. Therefore test chemical was considered to benot mutagenic in mouse lymphoma byin 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 the various test chemicals was reviewed to determine the mutagenic nature of 5-methoxy-1,3-dihydro-2H-benzimidazole-2-thione( 37052-78-1). The studies are as mentioned below:

AMES test

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether (CAS no. 93-04-9) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

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 compound using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs.  The test compound was used at a dosage level of 0, 3.3, 10.0, 33.0, 100.0, 200.0, 333.0, 667.0, 1000.0, 3333.0, 6666.0, 6667.0, 10000.0 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study.

 

Test substance did not induce a reproducible, dose-related increase in his+revertants over the corresponding solvent in the S. typhimurium tester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

In vitro chromosome abbreviation assay

 

In vitro chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Human peripheral blood lymphocytes with and without S9 metabolic activation system. The test chemical was dissolved in distilled water and used at dose level of0, 0.33, 1.0, 3.3, 10 mM. Concurrent solvent and positive control plates were also included in the study. The test chemical did not induce chromosome aberrations in Human peripheral blood lymphocytes with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

 

In another study, an in vitro mammalian cell gene Mutation test was carried out according to OECD guideline 476 using Chinese hamster ovary cells to evaluate the genetic potential of the test chemical. In results, The test chemical did not induced any chromosomal aberrations at concentrations up to 150-1000 μg/plate and 50-1000 μg/plate in Chinese hamster ovary cells 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

 

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells.

 

In the genotoxicity test, test substance was administered to CHO cells for 3 hrs at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 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 asN-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.

 

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 was seen in one well out of four at the concentration of 0.5 mM and in the absence with 4% S9 liver microsomal fraction, and two very diffuse colonies were detected in one well out of four at the concentration of 0.1 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the three spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present.

 

When the mutation frequency was determined, a frequency of 5.35 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 methyl 2-napthyl ether in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs.

 

Conclusion

Based on the results of the current study, we conclude that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs, in the presence or absence of metabolic activation.

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian cell gene mutation was performed .The test material was exposed tomouse lymphomaL5178Y cells. The concentration of test material used in the concentration of 0- 100 µg/mL. No mutagenic frequency were observed in the mammalian cells in presence and absence of metabolic activation. Therefore test chemical was considered to benot mutagenic in mouse lymphoma byin vitro mammalian cell gene mutation. Hence the substance cannot be classified as mutagenic in vitro.

 

Based on the data summarized, 5-methoxy-1,3-dihydro-2H-benzimidazole-2-thione( 37052-78-1). did not induce gene mutation .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 the test chemical5-methoxy-1,3-dihydro-2H-benzimidazole-2-thione( 37052-78-1). did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.