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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance CBS was negative in bacterial gene mutation assays (Monsanto Co. 1976, MHWJ 1997); also a mouse lymphoma assay was negative (Monsanto Co. 1979). An in vitro chromosomal aberration assay test gave weak evidence for a clastogenic potential in presence of metabolic activation, but the positive findings was assessed to be ambiguous. The only in vivo test (on embryonic mortality) cannot be assessed adequately due to insufficient data reporting. Based on the above discussed findings, it is assessed that CBS is not genotoxic, which is in line with the EU risk assessment.
It is stated in the EU risk assessment (2007) that no relevant evidence for mutagenicity of CBS and its hydrolysis products MBT and CHA was found.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
N-cyclohexylbenzothiazole-2-sulphenamide was tested in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
ames assay
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
6.25 - 5000 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acylamide (TA 98; TA 100);  sodium azide (TA 1535); 9-aminoacridine (TA 1537). with S9: 2-aminoanthracene (all strains)
Key result
Species / strain:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 100 µg/plate (TA1537 without S9); 500 µg/plate (TA 1537 with S9); 1500 µg/plate (TA 100 with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

No cytotoxicity was observed in TA 100 without S9 as well as in TA 98 and TA 1535, respectively.

Cytotoxicity: 100 µg/plate (TA1537 without S9); 500 µg/plate (TA 1537 with S9); 1500 µg/plate (TA 100 with S9). Precipitation of the test substance was noted at concentrations of 200 µg/plate and higher.

Conclusions:
Interpretation of results: negative
Executive summary:

The test substance CBS was evaluated in a GLP- and guideline study (OECD TG 471) bacterial gene mutation test. Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used. Treatment by the pre-incubation method was done for doses up to 5000 µg/plate. Toxicity effects varied in a strain-specific manner; at high doses from 200 µg/plate upward precipitations were observed. The test substance CBS was negative in presence and absence of metabolic activation (MHWJ 1997).

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:
other: GLP and guideline study, but with restriction concerning cytotoxicity determination and data documentation (study in jananese, abstract and tables in english available, no historical control data documented)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Genotoxicity of N-cyclohexyl-2-benzothiazolesulfenamide was studied by chromosomal aberration in cultured hamster lung (CHL/IU) cells.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosomal aberrations
Species / strain / cell type:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
10 - 81 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: mitomycin C (-S9), CPA (+S9)
Details on test system and experimental conditions:
CHL/IU cells.
Continuous treatment (24 and 48 h) without S9
Short term treatment (6 h) with and without S9(18 h sampling time)
Solvent: DMSO
Positive controls:
without S9: Mitomycin C
with S9: Cyclophosphamide
S9 from rat liver induced with phenobarbital and 5,6-benzoflavone.
2 plates per test.
Statistics:
Fisher's exact test using a Bonferroni's correction for multiple comparisions; Armitage's trend test.
Key result
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Cytotoxicity:
80 µg/ml (24 - 48 h treatment -S9)
83 µg/ml (6 h treatment -S9)
160 µg/ml (6 h Treatment +S9).

After 6 h treatment + S9 there were 4.5 % structural chromosomal  aberrations (incl. gaps) induced at 41 µg/ml and 1.88 % polyploidy at 81 µg/ml.

However a trend test showed no dose dependency for the structural chromosomal aberrations. In a confirmation test, 3.5 % structural chromosomal aberrations (incl.  gaps) were induced at 81 µg/ml and 1.13 % polyploidy at 41 µg/ml.

However a trend test showed no dose dependency for the structural chromosomal aberrations or the polyploidy.

Experiment I and II with metabolic activation, 6 h treatment

 Group  concentration µg/ml  TAG (%)  TA (%)  Polyploid (%)
 experiment I    
 Solvent (DMSO)  0  1 (0.5) 1 (0.5)   0.38
 CBS  41  9* (4.5)  7 (3.5)  0.88
 CBS  81 5 (2.5)  4 (2.0)  1.88*
 experiment II        
 Solvent (DMSO)  0  2 (1.0)  2 (1.0)  0.88
 CBS  20  4 (2.0)  4 (2.0)  0.38
 CBS  41  2 (1.0)  2 (1.0)  1.13*
 CBS  81  7* (3.5)  7 (3.5)  0.79

TAG: total no. of cells with aberrations

TA: total no. of cells with aberrations except gap

*:Significant different from histroical solvent control (p<0.05) by fisher's exact test using a Bonferroni's correction for multiple comparisions.

The statistically significant increase in aberrant cells and polyploidy after treatment with CBS with metabolic activation showed, when trend tested no dose dependency. In addition, according to recommendation given in the current OECD guideline (TG 473), the in vitro chromosome aberration assay is not appropriate method for the detection of polyploidy.

Conclusions:
Interpretation of results: ambiguous with metabolic activation weak positive with metabolic activation.
Executive summary:

The test substance CBS was weak positive in an in vitro chromosomal aberration using CHL cells (MHWJ 1997). In the absence of metabolic activation (S-9 mix) no induction of structural chromosomal aberrations was found for doses ranging from 10 to 41 µg/ml; no strong cytotoxicity was induced, but higher doses led to total cytotoxicity; various treatment and sampling times used (24/24 h; 48/48 h and 6/18 h). In the presence of S-9 mix weakly increased frequencies of structural chromosome aberration were found in two experiments after 6 hour treatment and 18 hours sampling. Again, no strong cytotoxicity was induced but higher doses led to total cytotoxicity. The main results were as followed: experiment I: 41 µg/ml, 81 µg/ml results in 3.5 % and 2.0% aberrant cells (excluded gaps); experiment II: 41 mg/ml, 81 µg/ml results in 1.0% and 3.5 % aberrant cells (excluded gaps). Since all solvent controls ranged from 0.5 to 1.0 aberrant cells (excluded gaps), this is interpreted as weak evidence for a clastogenic potential.

However, statistically evaluation revealed no dose dependency for structural chromosomal aberration. In addition, cytotoxcity was indicated in the first experiment as relative methaphase frequency to the solvent control and in the second experiment as cell confluency. No dose dependency in cytotoxicity was seen up to 0.081 mg/ml test substance, whereas higher concentrations could not be evaluated because of very high cytotoxcity. Thus, no dose-depencency in genotoxcity and cytotoxity was noted in the relevant concentration range. The increase in aberrant cells (3.5 %) is rather weak. In addition, the background frequency was low in both experiments (0.5% and 1%). compared to data from literature (XX). In addition, the slight increase in aberrant cells was stated to be significant different from historical solvent control data, but no historical control data are documented. Based on the rather weak increase in aberrant cells, the lack of a dose response relationship in genotoxcity and cytotoxcity and the low background frequency noted, the findings was assumed to be ambiguous.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: comparable to guideline study with acceptable restriction (e.g. positive controls not consistent with recent guideline, no differentiation on colony-size)
Principles of method if other than guideline:
Santocure BO-78-221 (N-cyclohexyl-2-benzothiazolesulfenamide) was tested for mutagenicity in a MLA at the TK locus in L5178Y mouse lymphoma cells with or without S9 metabolic activation according to the protocol of Clive, D., Mutation Res., 31, 17-29, 1975).
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Tk locus
Species / strain / cell type:
other: L5178Y mouse lymphoma assay
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.078 µg/ml to 40 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: EMS (-S9), dimethylnitrosamine (+S9)
Details on test system and experimental conditions:
Mouse lymphoma assay
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Cytotoxicity:
-S9:
No survivors at 39 µg/ml, 5.7% to 13.3% relative growth for the concentration range from 15 to 30 µg/ml.
+S9:
No survivors at 80 µg/ml, 75.2% relative growth at 40 µg/ml, toxicity was moderate to high in 0.313 to 20 µg/ml concentration range.

The test substance CBS did not induce a significant increase in mutations at the TK locus in L5178Y mouse lymphoma cells with or without S9 metabolic activation. A range of concentrations up to the highly toxic levels of 30 µg/ml (without metabolic activation) and 75 µg/ml (with metabolic activation) were assayed. No differentiation on colony-size was made.

The test substance CBS is considered to be inactive in the mouse lymphoma forward mutation assay.

Conclusions:
Interpretation of results: negative
Executive summary:

The test substance CBS was negative in a mouse lymphoma assay with L5178Y cells, with S-9 mix up to a dose of 75 µg /ml and without S9-mix up to a dose of 30 µg/ ml; toxic effects were found with and without S-9 mix in high doses (Monsanto Co 1979).

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

Genetic toxicity in vivo

Description of key information

The only in vivo test (on embryonic mortality) cannot be assessed adequately due to insufficient data reporting.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro data

The test substance CBS was evaluated in a GLP- and guideline study (OECD TK 471) bacterial gene mutation test. Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used. Treatment by the pre-incubation method was done for doses up to 5000 µg/plate. Toxicity effects varied in a strain-specific manner; at high doses from 200 µg/plate upward precipitations were observed. The test substance CBS was negative in presence and absence of metabolic activation (MHWJ 1997). These findings were confirmed by another Ames assay, employing Salmonella typhimurium tester strain TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Saccharomyces cerevisiae strain D4 with and without metabolic activation for doses ranging from 0.1 to 500 µg/ plate (Monsanto Co 1976).

The findings from the bacterial mutation assays were confirmed in a mammalian cell mutation assay. The test substance CBS was negative in a mouse lymphoma assay with L5178Y cells, with S-9 mix up to a dose of 75 µg /ml and without S9-mix up to a dose of 30 µg/ ml; toxic effects were found with and without S-9 mix in high doses (Monsanto Co 1979). No differentiation on colony-size was made.

However, the test substance CBS was weak positive in an in vitro chromosomal aberration using CHL cells. In the absence of metabolic activation (S-9 mix) no induction of structural chromosomal aberrations was found for doses ranging from 10 to 41 µg/ml; no strong cytotoxicity was induced, but higher doses led to total cytotoxicity; various treatment and sampling times used (24/24 h; 48/48 h and 6/18 h). In the presence of S-9 mix weakly increased frequencies of structural chromosome aberration were found in two experiments after 6 hour treatment and 18 hours sampling. Again, no strong cytotoxicity was induced but higher doses led to total cytotoxicity.

The main results were as followed: experiment I: 41 µg/ml, 81 µg/ml results in 3.5 % and 2.0% aberrant cells (excluded gaps); experiment II: 41 mg/ml, 81 µg/ml results in 1.0% and 3.5 % aberrant cells (excluded gaps).

Since all solvent controls ranged from 0.5 to 1.0 aberrant cells (excluded gaps), this is interpreted as weak evidence for a clastogenic potential. However, the author stated, that trend tests showed no dose dependency for structural chromosomal aberrations (MHWJ 1997).

In vivo data

Embryonic mortality of CBS was investigated in rats. However, no reliable data on dominant lethal mutations can be derived because test method and data presentation are inadequate (Aleksandrov 1982).


Short description of key information:
The test substance CBS was negative in bacterial gene mutation assays (Monsanto Co. 1976, MHWJ 1997); also a mouse lymphoma assay was negative (Monsanto Co. 1979). An in vitro chromosomal aberration assay test gave weak evidence for a clastogenic potential in presence of metabolic activation, but the positive findings was assessed to be ambiguous. The only in vivo test (on embryonic mortality) cannot be assessed adequately due to insufficient data reporting. Based on the above discussed findings, it is assessed that CBS is not genotoxic, which is in line with the EU risk assessment.
It is stated in the EU risk assessment (2007) that no relevant evidence for mutagenicity of CBS and its hydrolysis products MBT and CHA was found.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance CBS was negative in bacterial gene mutation assays (Monsanto Co. 1976, MHWJ 1997); also a mouse lymphoma assay was negative (Monsanto Co. 1979). An in vitro chromosomal aberration assay test gave weak evidence for a clastogenic potential in presence of metabolic activation, but the positive findings was assessed to be ambiguous. The only in vivo test (on embryonic mortality) cannot be assessed adequately due to insufficient data reporting. Based on the above discussed findings, it is assessed that CBS is not genotoxic, which is in line with the EU risk assessment.
It is stated in the EU risk assessment (2007) that no relevant evidence for mutagenicity of CBS and its hydrolysis products MBT and CHA was found.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.