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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In order to evaluate the genetic toxicity potential of the registered substance three in vitro tests according to OECD guidelines were performed: i.e. Ames test (OECD 471), Chromosome aberration test (OECD 476) and HGPRT (OECD 473) test. All of these three in vitro tests were negative in result.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-02-12 to 1999-03-16
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: in accordance with "Standards for Toxicity Investigations" .
Version / remarks:
Ministry of Labor, Notification No.77, September'l, 1988 and Notification No.67, June 2, 1997
GLP compliance:
yes
Remarks:
This study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988).
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Dose finding test: with and without S9 mix: 4.88, 19.5, 78.1, 313, 1250, 5000 (µg/plate)
Main test with and without S9 mix: 313, 625, 1250, 5000 (µg/plate)
Confirmation test: with strain TA1537 with and without S9 mix: 313, 625, 1250, 5000 (µg/plate)

Positive controls without S9 mix:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate (TA100, WP2uvrA), 0.1 µg/plate (TA98)
NaN3: Sodium azide: 0.5 µg/plate (TA1535)
ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropy1amino]acridine' 2HCl: 1 µg/plate (TA 1537)

Positive controls with S9 mix:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate (TA100, WP2uvrA), 0.1 µg/plate (TA98)
NaN3: Sodium azide: 0.5 µg/plate (TA1535)
ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropy1amino]acridine' 2HCl: 1 µg/plate (TA 1537)
2AA: 2-Aminoanthracene: 0.5 µg/plate (TA98), 1 µg/plate (TA100), 2 µg/plate (TA1535, TA1537), 10 µg/plate (WP2uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Since the solubility of the test substance was less than 50 mg/mL in water but more than 50 mg/mL in DMSO, DMSO was selected as a solvent. It was confirmed that there was no change in the 50 mg/mL solution in DMSO, including color change and heat generation, until 2 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The solvent used in the tests was employed as a negative control
Positive controls:
yes
Positive control substance:
sodium azide
other: 2(2-Furyl)3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
This study was performed by the pre-incubation method with and without S9 mix. Triplicate plates were used for the negative control group and duplicate plates per dose for the test substance treatment groups and the positive control groups. The test code number, name of test strain, presence or absence of S9 mix and dose level were noted on each plate.
Evaluation criteria:
The test substance was judged to be positive when the number of revertant colonies of the test substance treatment groups increased to twice or more that in the negative control with a concentration-dependent manner.
Statistics:
Any statistical methods were not used.
Key result
Species / strain:
other: TA 1535, TA 98, TA 100
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:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Result of Confirmation test: Number of revertant colonies in TA1537 strain was less than twice than in the negative control (with and without S9 mix). Increase of numbers of revertants in main test is juded accidential as value of negative group was low.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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:
valid
Additional information on results:
Confirmation test:
The number of revertant colonies in the test substance treatment groups was less than twice than in the negative control and in the groups with and without S9 mix in TA1537. Therefore, the increase of the number of revertant colonies in the main test was not confirmed dose-dependend and reproducible and due to the value of negative controls were low in the main test it was judged as an accidental increase result. The number of revertant colonies of the test substance treatment group in the main test was within the range of the historical data. From the above results, mutagenisity was judged to be negative in TA1537.

Remarks on result:
other: strain/cell type: salmonella typhimurium
Remarks:
Migrated from field 'Test system'.

Table 1: Summary of mean number of revertants in Salmonella typhimurium strains and E. coli WP2uvrA strain with and without metabolic activation (mean of 3 plates for vehicle controls and 2 plates for test item treated or positive controls)

Results of dose finding Test

Concentration

TA100

TA1535

WP2uvrA

TA98

TA1537

[µg/plate]

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative Control

159

131

7

8

19

19

19

28

4

9

4.88

137

140

6

7

20

25

18

37

3

13

19.5

131

143

7

6

17

27

17

29

3

10

78.1

131

141

13

11

17

23

17

24

3

7

313

111

136

6

10

18

28

20

28

3

10

1250

117

113

10

5

19

22

21

25

4

13

5000

104

97

7

6

15

32

20

21

4

11

positive Control

AF-2

2AA

NaN3

2AA

AF-2

2AA

AF-2

2AA

ICR-191

2AA

448

718

328

147

155

1050

585

291

2778

97

Results of Main Test

Concentration

TA100

TA1535

WP2uvrA

TA98

TA1537

[µg/plate]

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative Control

133

119

9

7

17

26

18

28

3

5

313

116

122

9

9

18

30

17

32

7

10

625

111

121

11

6

19

25

17

26

5

13

1250

106

114

7

7

21

23

19

24

8

17

2500

102

111

6

7

15

23

19

27

7

12

5000

92

91

5

7

19

23

20

20

6

11

positive Control

AF-2

2AA

NaN3

2AA

AF-2

2AA

AF-2

2AA

ICR-191

2AA

449

766

386

163

149

1046

554

394

2599

99

AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide = positive control –S9 mix for strains TA100, WP2uvrA and TA98 [selected doses of 0.01, 0.01 and 0.1 µg/plate, respectively]

NaN3: Sodium azide= positive control –S9 mix for strain TA1535 [selected dose:0.5 µg/plate]

ICR- 191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl = positive control –S9 mix for strain TA1537 [selected dose:1 µg/plate]

2AA: 2-Aminoanthracene = positive control + S9 mix for all tested bacteria strains[selected doses of 1, 2, 10, 0.5 and 2 µg/plate for strain TA100, TA1535, WP2uvrA, TA98 and TA1537, respectively]

 

Table 2: Confirmation Test: Summary of mean number of revertants in Salmonella typhimurium strain TA1537 with and without metabolic activation (mean of 3 plates for vehicle controls and 2 plates for test item treated or positive controls)

Results of confirmation Test

Concentration

TA1537

[µg/plate]

- S9

+ S9

Negative Control

8

10

313

10

10

625

9

11

1250

7

11

2500

10

11

5000

8

11

positive Control

 

ICR-191

2AA

2753

110

ICR- 191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine'2HCl [Dose:1 µg/plate]

2AA: 2-Aminoanthracene[Dose: 2 µg/plate]

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the above results, it is concluded that the registered substance has no ability to induce mutation under the conditions of the present study.
Executive summary:

The ability of registered substance to induce mutations was investigated using Salmonella typhimurium strains TAl00, TA1535, TA98 and TAl537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix).

As a result, increase in the numbers of revertant colonies was not observed at any doses of the test substance treatment group in all test strains with and without S9 mix.

The numbers of revertant colonies in the negative and positive controls were confirmed to be within the range of the historical data in the testing facility. Based on the above results, it is concluded that the registered substance has no ability to induce mutation under the conditions of the present study.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-22 to 2015-12-15
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.
Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on 08 August 2014.
Cells were grown in tissue culture flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate was used as the metabolic activation system
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 20, 40, 80, 160, 320, 640, 1280 and 2300 µg/mL
Initial gene mutation assay:
Experiment 1: 3-hour exposure in the Presence of Metabolic Activation: 57, 180, 570 and 1800 µg/mL
Experiment 2: 3-hour exposure in the Absence of Metabolic Activation: 56, 167, 500 and 1500 µg/mL
Confirmatory gene mutation assay:
Experiment 3: 3-hour exposure in the Absence of Metabolic Activation: 48, 150, 475 and 1500 µg/mL
Experiment 4: 3-hour exposure in the Presence of Metabolic Activation: 48, 150, 475 and 1500 µg/mL
Note: Experiment 4 was carried out to generate additional data in the presence of metabolic activation.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Evaluation criteria:
There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency.
Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. A test item, for which the results do not meet the above criteria is considered non mutagenic in this system.
CRITERIA FOR DATA ACCEPTABILITY
The Cloning Efficiency of the vehicle controls should not be less than 60%.
The mean mutant frequency of the vehicle controls in each experiment should fall within a range of 0 to 20 mutants per 10-e6 clonable cells.
The positive controls must induce a statistically significant response.
Statistics:
A power transformation procedure (Snee and Irr, 1981) with which, the observed mutant frequency was transformed using the formula:
Y = (X + A) B
where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p ≤ 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p ≤ 0.05).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested up to 2300 µg/mL (equivalent to 10mM)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that the test item, (Benzothiazol-2-ylthio)acetic acid does not have the potential to induce gene mutation in CHO cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

In order to determine the potential of the test substance to induce gene mutations in either the presence or absence of metabolic activation system (S9mix) in mammalian cells, an OECD 476 test in Chinese Hamster Ovary (CHO) cell line was performed. The assay conditions fulfilled all the specifications as per the OECD 476 guideline. A preliminary cytotoxicity test was performed at concentrations od 20, 40, 80, 160, 320, 640, 1280 and 2300 µg/mL testsubstance. DMSO served as vehicle.

Since the test item exhibited cytotoxicity, a maximum of 1800 and 1500 µg/mL test concentration was exposed in the assay in the presence and absence of S9 mix, respectively. Similarly, all the acceptance criteria for a valid test were met.The Cloning Efficiency of the vehicle controls was more than 60%. The mean mutant frequency of the vehicle controls in each experiment was between 11.7 and 18.2 mutants per 106clonable cells. The positive controls induced a statistically significant response.

No evidence for the induction of gene mutation was observed in any of the concentrations of the test item either in the presence or in the absence of metabolic activation.

Taken together, the results of the initial and confirmatory assays indicated that the test item does not have the potential to induce gene mutation either in the presence or in the absence of metabolic activation. In each of these assays, the positive control chemicals produced a statistically significant increase in the mutant frequencies, demonstrating that the system was able to detect the effect of known mutagens.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-18 to 2015-12-15
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.
Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on 08 August 2014.
Cells were grown in tissue culture flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate was used as the metabolic activation system
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 20, 40, 80, 160, 320, 640, 1280 and 2300 µg/mL
Initial gene mutation assay:
Experiment 1: 3-hour exposure in the Presence of Metabolic Activation: 55, 158 and 500 µg/mL
Experiment 2: 3-hour exposure in the Absence of Metabolic Activation: 40, 126 and 400 µg/mL
Experiment 3: 21-hour exposure in the Absence of Metabolic Activation: 40, 126 and 400 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Evaluation criteria:
As a guide to interpretation of the data, the test item will be considered to induce a positive response when the percentage of cells with aberrations is increased in a dose responsive manner with one or more concentrations being statistically significant (p ≤ 0.05). If there is a statistically significant increase over the control in only one dose, to prove a reproducible increase, an independent confirmatory assay will be done.

For structural chromosome aberrations, values that are statistically significant but do not exceed the range of historic vehicle controls may be judged as non-biologically significant. Test items not demonstrating a statistically significant increase in aberrations will be considered as negative.

An increase in the number of polyploidy cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle progression.
The in vitro Mammalian Chromosome Aberration Test is considered acceptable, if it meets the following criteria:
1. The incidence of aberrations in the vehicle control cultures is in the range of in-house historical control data.
2. The positive control substances should produce a significant increase in the incidence of aberrations compared to the respective vehicle control.
Statistics:
Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations, numerical aberration (Poly) were analyzed. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fisher exact test. All analysis and comparisons were evaluated at 5 % (p ≤ 0.05) level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control values at all the concentrations tested with and without S9-mix activation after 3 h as well as 21 h exposure.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Test item showed required level of cytotoxicity (about 50% cytotoxicity cell growth inhibition compared to control) between 320 and 640 µg/mL with/without S9-mix activation with 3-h exposure as well as without S9-mix activation with 21h exposure.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It was concluded that the test item, (Benzothiazol-2-ylthio)acetic acid was not clastogenic in CHO cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

In order to determine the potential of the test substance to be clastogenic in either the presence or absence of metabolic activation system (S9 mix) in mammalian cells, a chromosome aberration test in Chinese Hamster Ovary (CHO) cell line was performed. The assay conditions fulfilled all the specifications as per the OECD 473 guideline.

A preliminary cytotoxicity test was performed at concentrations of 20, 40, 80, 160, 320, 640, 1280 and 2300 µg/mL with the test substance. DMSO served as vehicle. (Benzothiazol-2-ylthio)acetic acid showed required level of cytotoxicity (at least 50% cytotoxicity as cell growth inhibition compared to the vehicle control) between 320 and 640 µg/mL in the presence and absence of metabolic activation with 3-hour exposure as well as in the absence of metabolic activation with 21-hour exposure. Based on these observations, for the initial chromosome aberration assays, a maximum of 500 µg/mL and 400 µg/mL was tested in the presence and absence of metabolic activation with 3-hour exposure, respectively. Similarly, for the confirmatory test a maximum of 400 µg/mL was tested in the absence of metabolic activation with 21-hour exposure.

There was no evidence of an increase in either structural or numerical aberrations in cultures treated with (Benzothiazol-2-ylthio)acetic acid either in the presence or in the absence of metabolic activation.

The results of this study suggest that the test item, (Benzothiazol-2-ylthio)acetic acid does not have the potential to cause chromosome damage in the presence or absence of metabolic activation.

In each of these experiments, the respective positive controls produced a statistically significant increase in aberrant metaphases, demonstrating that the system was able to detect the effect of known mutagens.

It was concluded that the test item, (Benzothiazol-2-ylthio)acetic acid wasnot clastogenic in CHO cells at the tested concentrations and under the conditions of test employed.

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

Genetic toxicity in vivo

Description of key information

Results from three different in vitro test systems with the registered substance are available which are all negative in result. All these results do not indicate specific concern for carcinogenicity. Thus from a scientific sight of view and supported by animal welfare reasons additional in vivo testing is not necessary.

Additional information

Justification for classification or non-classification

In order to evaluate the genetic toxicity potential of the registered substance three in vitro tests according to OECD guidelines were performed: i.e. Ames test (OECD 471), Chromosome aberration test (OECD473) and HGPRT (OECD 476) test. All of these three in vitro tests were negative in result. According to Regulation (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, the test substance (Benzothiazol-2-ylthio)acetic acid has not to be classified.