Benzimidazole-2-thiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES assay:.

Test substance was tested for its genotoxic potetial in the Salmonella/microsome assay with preincubation using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation (S9 mix from Arocolor 1254-induced rat and Syrian hamster livers.). The substance was not likly to exibit gene mutations.

chromosome aberration study

No chromosomal structural abnormalities and induction of polyploid cells were observed in all groups treated for 24 hours with the addition of test substance. On the other hand, chromosome structural abnormality was observed in 7.0% (including gap) of observed cells in the high concentration group (0.39 mg / ml) continuously treated for 48 hours, and false positive results were obtained. No significant increase in chromosome structural abnormality and polyploid cells was observed in the other treatment group. No chromosomal structural abnormality and polyploid cell induction effect were observed in all treatment groups treated with test substance for 6 hours in the absence of S9 mix. On the other hand, a significant increase in structural abnormality of chromosomes was observed in all treatment groups in the presence of S9 mix, and the frequency of appearance was 4.0 to 12.5% ​​(including gap), and the judgment was positive. In addition, significant increase of ploidy cells was observed in all treatment groups in the presence of S9 mix, but the frequency of occurrence was 4.00 to 4.75%.Thus test substance is not likly to clssified as gene mutant.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Preincubation Assay
The preincubation assay was performed as described previously [Haworth et al,1983]. 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 and TA 100

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:

S9 mix from Arocolor 1254-induced rat and Syrian hamster livers.

Test concentrations with justification for top dose:

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 chemical was soluble 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

Remarks:

For strains TA1535 and TA 100 in the absence of metabolic activation.

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

Remarks:

For strain TA 1537 in the absence of metabolic activation.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamin

Remarks:

For strain TA98 in the absence of metabolic activation.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of metabolic activation for all strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- 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
- 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: Triplicate

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:

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

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 distinctions between a weak mutagenic response and a mutagenic response, or between a weak mutagenic response and a questionable mutagenic response are highly subjective.

A 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. A chemical was considered to be questionable (?) if a reproducible increase of hist 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. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity .

Statistics:

Mean and SEM

Species / strain:

S. typhimurium 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 available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

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

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic potential was observed

Table: Mutagenicity of 2-Mercaptobenzimidazole (benzimidazole-2-thiol)

Dose (µg/plate)	TA100
	-S9		10% HLI		30% HLI		10% RLI		30%HLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	98	5.6	68	3.2	143	3.3	81	0.9	125	2.8
3
10
33.3	93	2.5			123	5.5			117	3.8
100.0	96	2.2	59	1.8	131	22.1	76	1.2	118	3.1
200
333.0	77	5.3	64	2.9	115	11.2	74	5.4	94	10.4
667.0
1000	57	0.7	63	2.0	122	16.7	69	3.5	92	6.2
3333	34s	2.3	62	5.2	84	20.2	39	2.3	69s	5.0
6666			14	2.0			14	2.2
6667					38	7.0			49s	15.6
10000
Positive control	1060	27.8	901	4.7	871	37.4	1415	31.7	598	19.5

 

Dose (µg/plate)	TA1535
	-S9		10% HLI		30% HLI		10% RLI		30%HLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	28	2.9	7	1.7	12	2.6	7	0.6	12	2.0
3
10
33.3	24	4.5
100.0	28	3.3	8	2.0	10	2.6	6	1.2	9	2.0
200
333.0	29	1.8	7	1.3	10	0.9	8	2.4	9	1.3
667.0
1000	40	0.9	8	2.0	7	2.3	7	0.7	5	1.2
3333	35	4.3	6	0.9	5	0.7	4	0.6	5	2.0
6666			5	0.9	3	0.3	3	0.3	3	0.7
6667
10000
Positive control	1009	33.1	119	10.6	119	10.8	114	7.7	96	4.3

 

Dose (µg/plate)	TA97
	-S9		10% HLI		30% HLI		10% RLI		30%HLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	100	6.9	86	1.8	194	6.9	84	3.5	167	3.2
3	106	7.7
10	102	6.5			182	11.1			174	4.4
33.3	94	8.8			189	8.3			149	17.9
100.0	86	2.3	80	2.0	188	6.7	83	1.9	167	4.4
200	75	1.8
333.0			73	4.3	152	9.7	72	1.7	110	3.8
667.0					58	8.0			96	7.2
1000			16	6.1			8	1.5
3333			20	3.4			8	1.5
6666			2	1.2			1	0.6
6667
10000
Positive control	656	7.0	910	11.9	839	38.1	1402	4204	557	2.3

 

Dose (µg/plate)	TA98
	-S9		10% HLI		30% HLI		10% RLI		30%HLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	24	3.9	21	2.2	34	0.9	71	3.7	21	3.2
3
10
33.3	20	3.2
100.0	12	1.2	19	2.8	32	0.9	26	3.3
200
333.0	14	3.8	25	4.8	37	0.7	24	1.0	24	0.0
667.0
1000	17	1.2	20	3.2	33	4.6	22	4.4	23	3.8
3333	14	1.0	23	1.5	50	4.8	19	3.1	20	5.5
6666			25	5.0			23	2.3
6667					44	4.7			27	5.8
10000					32	1.5			24	2.6
Positive control	2029	18.0	823	20.7	90	0.0	1328	40.0	474	0.6

 

s: slight clearing of background material

Conclusions:

Test substance was tested for its genotoxic potetial in the Salmonella/microsome assay with preincubation using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation (S9 mix from Arocolor 1254-induced rat and Syrian hamster livers.). The substance was not likly to exibit gene mutations.

Executive summary:

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 substance 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 J- check

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

To evaluate the cytogenetic effect on cultured cells of test substance as a part of toxicity study survey project on existing chemical safety inspection, test using Chinese hamster cultured cells (CHL / IU) Tube in vitro chromosome aberration test was carried out.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Details on mammalian cell type (if applicable):

- Type and identity of media including CO2 concentration if applicable: Eagle MEM culture broth supplemented with 10% fetal bovine serum
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 (Rat liver, induced with phenobarbital and 5,6-benzoflavone)

Test concentrations with justification for top dose:

-S9 (continuous treatment): 0, 0.10, 0.20, 0.39 mg/ml
-S9 (short-term treatment): 0, 0.4, 0.8, 1.5 mg/ml
+S9 (short-term treatment): 0, 0.4, 0.8, 1.5 mg/ml

Vehicle / solvent:

Solvent : Dimethyl sulfoxide - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test chemical was soluble 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:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Continuous treatment: 24 and 48 hrs
Short term treatment: 6 hrs

- Expression time (cells in growth medium): Continuous treatment: 24 and 48 hrs
Short term treatment: 6 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): Colcemid

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: 2 plates/test

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Chromosome specimens were prepared according to a conventional method. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution.

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: Yes
- 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:

Chromosomes were analyzed based on the classification method by the Japan Society of Environmental Mutagenesis, Mammalian Examination (MMS) Subcommittee, and the presence or absence of structural abnormality such as chromosome type or chromosome type gap, cutting, exchange, The presence or absence of cells (polyploid) was observed. Regarding structural abnormalities, the mid-metaphase cells of 200 groups per group and 800 groups of group cells for ploidy cells.

Statistics:

A significant difference test (p <0.05) between the solvent control group and the test substance treated group and between the solvent control group and the positive control group was performed on the frequency of occurrence of cells having chromosomal abnormality by Fisher's exact probability test method.

According to the criteria of Ishikan et al., the frequency of chromosomal abnormalities is negative in less than 5%, false positives in less than 5% and less than 10%, more than 10% as positive.

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Remarks:

Continuous treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Remarks:

Short term treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Remarks:

Short term treatment

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. The proliferation inhibitory effect of the test substance on CHL / IU cells was determined by measuring the proliferation of each group using a monolayer culture cell densitometer and the solvent control group was used as an index.

As a result, the concentration clearly exceeding the 50% proliferation inhibitory concentration in continuous treatment (about 60% proliferation inhibitory concentration) was calculated from the value of 2 concentration sandwiching the proliferation inhibitory concentration of 60%, and it was 0.39 mg / ml there were. On the other hand, in the presence and absence of S9 mix for a short time treatment, growth inhibition clearly exceeding 50% was not observed in all treated concentration ranges

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: At the highest concentration group (0.39 mg/ml) with the 48-hr continuous treatment, cells with structural chromosomal aberrations including gap were marginally induced (7%). However the results were false positive.

Table: Chromosome analysis of Chinese Hamster cells (CHL/IU) continuously treated with the test material without S9 mix

Group	Dose (mg/mL)	Time of exposure (h)	No. of cells analayzed	No. of structural aberrations								Others	No. cells with aberrations		Polyploid (%)	Judgement
				Gap	Ctb	Cte	Csb	cse	F	Mul	total		TAG	TA		SA	NA
Control			200	1	0	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)	0.13
Solvent	0	24	200	2	1	0	0	0	0	0	3	1	3 (1.5)	1 (0.5)	0.38
MBI	0.10	24	200	1	0	0	0	0	2	0	3	0	3 (1.5)	2 (1.0)	0.00	-	-
MBI	0.20	24	200	1	2	2	0	1	0	0	6	0	5 (2.5)	4 (2.0)	0.13	-	-
MBI	0.39	24	200	0	1	0	0	0	1	10	12	0	3 (1.5)	3 (1.5)	0.00	-	-
MC	0.00005	24	200	7	98	138	6	3	4	0	256	3	123* (61.5)	120* (60.0)	0.00	+	-
Solvent	0	48	200	1	1	0	1	0	1	0	4	0	4 (2.0)	3 (1.5)	0.13
MBI	0.10	48	200	0	0	0	0	0	0	0	0	1	0 (0.0)	0 (0.0)	0.25	-	-
MBI	0.20	48	200	0	2	2	0	0	0	0	4	0	1 (0.5)	1 (0.5)	0.13	-	-
MBI	0.39	48	200	0	20	5	2	0	1	10	38	0	14* (7.0)	14* (7.0)	0.85	±	-
MC	0.00005	48	200	4	76	154	6	3	1	70	314	28	119* (59.5)	118* (59.0)	0.38	+	-

 

Table: Chromosome analysis of Chinese Hamster cells (CHL/IU) continuously treated with the test material with and without S9 mix

Group	Dose (mg/mL)	Time of exposure (h)	No. of cells analayzed	No. of structural aberrations								Others	No. cells with aberrations		Polyploid (%)	Judgement
				Gap	Ctb	Cte	Csb	cse	F	Mul	total		TAG	TA		SA	NA
Control			200	1	0	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)	0.00
Solvent	0	6- (18)	200	0	0	0	0	0	1	0	1	0	1 (0.5)	1 (0.5)	0.13
MBI	0.4	6- (18)	200	0	1	4	0	0	0	10	15	0	2 (1.0)	2 (1.0)	0.38	-	-
MBI	0.8	6- (18)	200	0	0	1	5	0	1	0	7	0	1 (0.5)	1 (0.5)	0.25	-	-
MBI	1.5	6- (18)	200	0	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.13	-	-
MC	0.005	6- (18)	200	0	0	0	0	0	1	0	1	1	1 (0.5)	1 (0.5)	0.00	+	-
Solvent	0	6- (18)	200	0	0	1	0	0	0	0	1	0	1 (0.5)	1 (0.5)	0.25
MBI	0.4	6- (18)	200	2	3	14	1	0	0	0	20	0	8* (4.0)	7* (3.5)	4.25	-	-
MBI	0.8	6- (18)	200	2	11	2/8	1	0	0	10	52	1	23* (11.5)	22* (11.0)	4.00	-	-
MBI	1.5	6- (18)	200	3	21	24	0	0	0	0	48	0	25* (12.5)	23* (11.5)	4.75	±	-
MC	0.005	6- (18)	200	5	158	267	12	2	3	200	647	2	159* (79.5)	158* (79.0)	0.13	+	-

 

Conclusions:

No chromosomal structural abnormalities and induction of polyploid cells were observed in all groups treated for 24 hours with the addition of test substance. On the other hand, chromosome structural abnormality was observed in 7.0% (including gap) of observed cells in the high concentration group (0.39 mg / ml) continuously treated for 48 hours, and false positive results were obtained. No significant increase in chromosome structural abnormality and polyploid cells was observed in the other treatment group.

No chromosomal structural abnormality and polyploid cell induction effect were observed in all treatment groups treated with test substance for 6 hours in the absence of S9 mix. On the other hand, a significant increase in structural abnormality of chromosomes was observed in all treatment groups in the presence of S9 mix, and the frequency of appearance was 4.0 to 12.5% ​​(including gap), and the judgment was positive. In addition, significant increase of ploidy cells was observed in all treatment groups in the presence of S9 mix, but the frequency of occurrence was 4.00 to 4.75%.

Executive summary:

To evaluate the cytogenetic effect on cultured cells of test substance as a part of toxicity study survey project on existing chemical safety inspection, test using Chinese hamster cultured cells (CHL / IU) Tube in vitro chromosome aberration test was carried out.

In the continuous treatment (24 and 48 hours), the maximum treatment concentration was defined as a growth inhibition concentration exceeding 50%, that is, a concentration of 0.39 mg / ml. On the other hand, since the growth suppression exceeding 50% was not observed in the presence or absence of S9 mix for a short time treatment (6 hours), the concentration of 1.5 mg / ml (10 mM) was taken as the maximum treatment concentration . 1/2 and 1/4 of the maximum treatment concentration were set as medium and low concentrations, respectively. In the continuous treatment, specimens were prepared after continuous treatment for 24 hours and 48 hours in the absence of S9 mix, after 6 hours treatment (18 hour recovery time) in the presence and absence of S9 mix for short time treatment, Chromosome abnormality induction was examined by mirroring.

No chromosomal structural abnormality or polyploid cell inducing effect was observed in any treatment group treated CHL / IU cells for 24 hours continuously. On the other hand, chromosome structural abnormality was observed in 7.0% (including gap) of observed cells in the high concentration group (0.39 mg / ml) continuously treated for 48 hours, and false positive results were obtained. In other treatment groups, chromosome structural abnormality and polyploid cell inducing action were not observed.

In the short-term treatment, no chromosomal structural abnormality or polyploid cell induction effect was observed in all treatment groups treated for 6 hours in the absence of S9 mix. On the other hand, a significant increase in structural abnormality of chromosomes was observed in all treatment groups in the presence of S9 mix, and the frequency of appearance was 4.0 to 12.5%, and a positive result was obtained. In addition, significant increase of ploidy cells was observed in all treatment groups in the presence of S9 mix, but the frequency of occurrence was 4.00 to 4.75%.

From the above results, it was concluded that test substance does not induces chromosomal abnormalities in CHL / IU cells in vitro without S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Test substance did not induce an increase in the number of micronucleated polychromatic or normochromatic erythrocytes as compared with the controls.Thus, test substance was devoid of clastogenicity under these experimental conditions.Therefore, it is considered that test substance is non mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The rodent bone marrow erythrocyte micronucleus (MN) assay was performed to screen the test chemical in vivo for genotoxicity resulting from clastogenic activity or mitotic damage.

GLP compliance:

not specified

Type of assay:

other: The rodent bone marrow erythrocyte micronucleus (MN) assay

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

inhalation

Vehicle:

- Vehicle(s)/solvent(s) used: air

Details on exposure:

No data

Duration of treatment / exposure:

90days

Frequency of treatment:

6 hr/day, 5 day/week, over a period of 90 days.

Dose / conc.:

0 mg/m³ air

Dose / conc.:

12.5 mg/m³ air

Dose / conc.:

25 mg/m³ air

Dose / conc.:

50 mg/m³ air

No. of animals per sex per dose:

Total: 40
0.0 mg/m3: 10 male and 10 female mice
12.5 mg/m3: 10 male and 10 female mice
25.0 mg/m3: 10 male and 10 female mice
50.0 mg/m3: 10 male and 10 female mice

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Tissues and cell types examined:

Blood smear was observed for polychromatic and normochromatic eryhtrocytes and the micronucleus frequency was scores

Details of tissue and slide preparation:

METHOD OF ANALYSIS: Peripheral blood smears were prepared from B6C3F1 mice by the NTP contract laboratory conducting the toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. The micronucleus frequencies in blood obtained by all four of these methods are similar.

DETAILS OF SLIDE PREPARATION: Slides were stained and analyzed at the USDA Western Regional Laboratory, under the direction of Dr. James MacGregor.
Drops of blood were spread on precleaned standard glass microscope slides, air dried, and fixed in absolute methanol for 5 min. Slides stained with acridine orange or
Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy, and slides stained with Giemsa were scored. by Koehler microscopy at 1000X.

Evaluation criteria:

Criteria for identification of MN were those of Schmid [1976] with the additional requirement that MN exhibit the fluorescence emission characteristic of the fluorescent stain used (blue with UV excitation and orange with green [540nm] excitation with Hoechst/pyronin stain, or yellow to greenish yellow with acridine orange stain).

Polychromatic erythrocytes (PCE) were scored by direct manual counting.

Normochromatic erythrocytes (NCE) were scored using a semiautomated method described by Jauhar et al. [1988], in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 (USDA) erythrocytes.

Although statistical analyses were used as an important aid in evaluating the test results. A decision to classify a test as negative, equivocal, or positive for induction of micronuclei in this in vivo assay was based on a broader evaluation of a number of factors that determined the biological relevance of the results, including the appropriateness of the concurrent control data, the magnitude of the observed response and the presence of a dose-dependent increase in the frequency of micronucleated cells. Test chemicals were evaluated separately in each gender; results from tests in male and female mice were not combined to reach an overall conclusion. Positive control groups were not routinely included in these subchronic MN tests because the toxicity bioassays from which the test animals were obtained did not include positive control groups.

Statistics:

The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an unadjusted one-tailed Pearson x2 test that incorporated the calculated variance inflation factor for the study. Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative.

The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Systemic toxicity was observed in the female mice at all test concentrations and in the male mice at concentration levels of and above 6.25 mg/m3.

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The result of the micronucleus test carried out at the end of a 90-day inhalation study must be considered relevant in accordance with OECD guideline No. 474, as in this 90-day study systemic toxicity was observed in the female mice at all test concentrations and in the male mice at concentration levels of and above 6.25 mg/m3, and also because the mice received test substance treatment upto the time of sacrifice.

Conclusions:

Test substance did not induce an increase in the number of micronucleated polychromatic or normochromatic erythrocytes as compared with the controls.Thus, test substance was devoid of clastogenicity under these experimental conditions.Therefore, it is considered that test substance is non mutagenic.

Executive summary:

The rodent bone marrow erythrocyte micronucleus (MN) assay was performed to screen the test chemical in vivo for genotoxicity resulting from clastogenic activity or mitotic damage. The test chemical was dissolved in suitable solvent and used at dose levels of 0, 12.5, 25 or 50 mg/m³. In none of the groups treated with test substance were any of the male or female mice found to have increased numbers of micronucleated polychromatic or normochrom atic erythrocytes as compared with the controls.Thus, test substance was devoid of clastogenicity under these experimental conditions.Therefore, it is considered that the test substance is non mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of the given test chemical to determine its mutagenic nature in vitro. The studies are as mentioned below:

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 by Zeiger et al (Environmental mutagenesis, 1987) 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 another study mentioned test substance , a reversion mutation test using bacteria was performed by the plate method. In the dose setting test, both S9 Mix-free and addition tests were carried out at 50 to 5000 μg / plate using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP 2 uvr A. Since antibacterial activity was not observed, in this test, the test was carried out at doses of 0, 312.5, 625, 1250, 2500, 5000 μg / plate for both S9 Mix-free and addition tests. Concurrent solvent and positive control chemicals were included in the study. As a result, no increase in the number of mutant colonies which were more than twice the solvent control was observed in any of the five tested bacteria used at any of the two tests in the two tests, so that the test substance was judged as having no mutagenicity (negative) in the test system used. 2-mercaptobenzimidazole did not induce gene mutation in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP 2 uvr A in the presence and absence of S9 metabolic activation system and hence it is noty likely to classify as a gene mutant in vitro.

To evaluate the cytogenetic effect on cultured cells of test substance as a part of toxicity study survey project on existing chemical safety inspection, test using Chinese hamster cultured cells (CHL / IU) Tube in vitro chromosome aberration test was carried out (J check, 2017). In the continuous treatment (24 and 48 hours), the maximum treatment concentration was defined as a growth inhibition concentration exceeding 50%, that is, a concentration of 0.39 mg / ml. On the other hand, since the growth suppression exceeding 50% was not observed in the presence or absence of S9 mix for a short time treatment (6 hours), the concentration of 1.5 mg / ml (10 mM) was taken as the maximum treatment concentration . 1/2 and 1/4 of the maximum treatment concentration were set as medium and low concentrations, respectively. In the continuous treatment, specimens were prepared after continuous treatment for 24 hours and 48 hours in the absence of S9 mix, after 6 hours treatment (18 hour recovery time) in the presence and absence of S9 mix for short time treatment, Chromosome abnormality induction was examined by mirroring. No chromosomal structural abnormality or polyploid cell inducing effect was observed in any treatment group treated CHL / IU cells for 24 hours continuous ly. On the other hand, chromosome structural abnormality was observed in 7.0% (including gap) of observed cells in the high concentration group (0.39 mg / ml) continuously treated for 48 hours, and false positive results were obtained. In other treatment groups, chromosome structural abnormality and polyploid cell inducing action were not observed. In the short-term treatment, no chromosomal structural abnormality or polyploid cell induction effect was observed in all treatment groups treated for 6 hours in the absence of S9 mix. On the other hand, a significant increase in structural abnormality of chromosomes was observed in all treatment groups in the presence of S9 mix, and the frequency of appearance was 4.0 to 12.5%, and a positive result was obtained. In addition, significant increase of ploidy cells was observed in all treatment groups in the presence of S9 mix, but the frequency of occurrence was 4.00 to 4.75%. From the above results, it was concluded that test substance does not induces chromosomal abnormalities in CHL / IU cells in vitro without S9 metabolic activation.

Test substance was tested for its genotoxic potetial in the Salmonella/microsome assay with plate incorporation test using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 (Secondary source: BG Chemie, 2000). 2-Mercaptobenzimidazole (benzimidazole-2-thiol) is devoid of any mutagenic potential both in the absence and presence of metabolic activation (S9 mix from Arocolor 1254-induced rat livers) and hence is not likely to classify as a gene mutant in vitro.

Genetic toxicity in vivo:

Various peer reviewed publications were reviewed to determine the mutagenic nature of the given test chemical to determine its mutagenic nature in vivo. The studies are as mentioned below:

The rodent bone marrow erythrocyte micronucleus (MN) assay was performed by WItt et al (Environmental and molecular mutagenesis, 2000) to screen the test chemical in vivo for genotoxicity resulting from clastogenic activity or mitotic damage. The test chemical was dissolved in suitable solvent and used at dose levels of 0, 12.5, 25 or 50 mg/m³. In none of the groups treated with test substance were any of the male or female mice found to have increased numbers of micronucleated polychromatic or normochrom atic erythrocytes as compared with the controls.Thus, test was devoid of clastogenicity under these experimental conditions.Therefore, it is considered that test substance is non mutagenic.

Sex linked recessive lethal mutation was carried out with test substance to investigate gene mutation toxicity in vivo using Drosophila melanogaster . The test chemical was tested ar dose levels of 2 -5 mg/mL. Only one out of a total of 963 investigated chromosomes was found to carry a lethal mutation. The lethal mutation rate of 2-mercaptobenzimidazole was 0.17% and hence lower than that of the incorporated control, which was 0.21% (6 out of 2869 lethal mutations). Thus 2-mercaptobe nzimidazole (benzimidazole-2-thiol) tested negative in this study.

Based on the data available for the target chemical, it does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo.

Justification for classification or non-classification

Based on the data available for the the test chemical, it does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo.