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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2011 - 01 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(piperidinothiocarbonyl) hexasulphide
EC Number:
213-537-2
EC Name:
Bis(piperidinothiocarbonyl) hexasulphide
Cas Number:
971-15-3
Molecular formula:
C12H20N2S8
IUPAC Name:
1,1'-(hexasulfane-1,6-diyldicarbonothioyl)dipiperidine
Constituent 2
Reference substance name:
Dipentamethylenethiuram hexasulfide
IUPAC Name:
Dipentamethylenethiuram hexasulfide
Test material form:
solid: particulate/powder

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
The dose-levels tested in the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Experiments without S9 mix
The selected treatment-levels were:
- 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains in the first experiment,
- 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/plate for the TA 100 strain in the first experiment,
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 98 and TA 102 strains in the second experiment,
- 3.9, 7.8, 15.6, 31.3, 32.5 and 125 µg/plate for the TA 1537 strain in the second experiment,
- 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/plate for the TA 100 strain in the second experiment.

Experiments with S9 mix
The selected treatment-levels were:
- 31.3, 62.5, 125, 250 and 500 µg/plate for the five strains in the first experiment,
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains in the second experiment,
- 7.8, 15.6, 31.3, 62.5, 125 and 250 µg/plate for the TA 100 strain in the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: In the solubility assay, the test item was found indissoluble in the vehicles usually used for this type of assay (water for injections, DMSO, acetone, and tetrahydrofuran). A suspension was therefore selected for the treatment. Since a homogenous suspension (checked with the naked eye) was obtained in DMSO at the concentration of 50 mg/mL (which enabled to test the highest dose-level recommended in the international guidelines), DMSO was selected as the vehicle in this study.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method.
The direct plate incorporation method was performed as follows: test item suspension (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 50°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The pre-incubation method was performed as follows: test item suspension (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37°C, the number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Also, the thinning of the bacterial lawn and the presence of precipitate were evaluated.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
No.
This study is considered valid if the following criteria are fully met:
. the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
. the number of revertants in the positive controls is higher than that of the vehicle controls (at least 2-fold increase (for the TA 98, TA 100 and TA 102 strains) and at least 3-fold increase (for the TA 1535 and TA 1537 strains)) and is consistent with the historical data of the testing facility.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
SOLUBILITY ASSAY AND PRELIMINARY TOXICITY TEST (Table 1)
In the solubility assay, the test item was found indissoluble in the vehicles usually used for this type of assay (water for injections, DMSO, acetone, and tetrahydrofuran). A suspension was therefore selected for the treatment. Since a homogenous suspension (checked with the naked-eye) was obtained in DMSO at the concentration of 50 mg/mL (which enabled to test the highest dose-level recommended in the international guidelines), DMSO was selected as the vehicle in this study.
The dose-levels tested in the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate. A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels = 500 µg/plate. This precipitate prevented the scoring of the plates at dose-levels = 2500 µg/plate. A moderate to strong toxicity was noted at dose-levels = 500 µg/plate in the TA 100 strain with and without S9 mix, = 2500 µg/plate in the TA 98 strain without S9 mix and at 5000 µg/plate in the TA 98 strain with S9 mix. No noteworthy toxicity was noted in the TA 102 strain, either with or without S9 mix.

MUTAGENICITY EXPERIMENTS (Tables 2 and 3)
The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered valid.
Since the test item was poorly soluble and sometimes cytotoxic in the preliminary test, the choice of the highest dose-level was based either on the level of toxicity or on the level of precipitate, according to the criteria specified in the international guidelines.

Experiments without S9 mix
A moderate precipitate was observed in the Petri plates when scoring the revertants at 500 µg/plate.
A moderate to strong toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at dose-levels = 15.6 µg/plate in the TA 100 strain, = 125 µg/plate in the TA 1537 strain and at 500 µg/plate in the TA 1535, TA 98 and TA 102 strains.
A noteworthy increase in the number of revertant colonies was noted in the TA 100 strain in the second experiment. This increase exceeded slightly the positive threshold of 2-fold the vehicle control value (2.2-fold), however, there was no evidence of a dose-response relationship and no similar effect was noted in the first experiment. Consequently, this increase was not considered as biologically relevant.
The test item did not induce any other noteworthy increase in the number of revertants.

Experiments with S9 mix
A moderate precipitate was observed in the Petri plates mainly at 500 µg/plate.
A moderate toxicity (thinning of the bacterial lawn) was noted at dose-levels = 125 µg/plate in the TA 1537 and TA 100 strains, = 250 µg/plate in the TA 1535 strain and at 500 µg/plate in the TA 102 strain.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

Applicant's summary and conclusion

Conclusions:
The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test item was suspended in dimethylsulfoxide (DMSO).

 

Results

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered valid.

 

Since the test item was poorly soluble and sometimes cytotoxic in the preliminary test, the choice of the highest dose-level was based either on the level of toxicity or on the level of precipitate, according to the criteria specified in the international guidelines.

 

A moderate precipitate was observed in the Petri plates when scoring the revertants at 500 µg/plate.

 

Experiments without S9 mix

The selected treatment-levels were ranged from 1.0 to 500 µg/plate.

A moderate to strong toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at dose-levels >= 15.6 µg/plate in the TA 100 strain, >= 125 µg/plate in the TA 1537 strain and at 500 µg/plate in the TA 1535, TA 98 and TA 102 strains.

No noteworthy increase in the number of revertants, which could be considered as biologically relevant, was noted in any of the five strains used.

Experiments with S9 mix

The selected treatment-levels were ranged from 7.8 to 500 µg/plate.

A moderate precipitate was observed in the Petri plates mainly at 500 µg/plate. 

A moderate toxicity (thinning of the bacterial lawn) was noted at dose-levels >= 125 µg/plate in the TA 1537 and TA 100 strains, >= 250 µg/plate in the TA 1535 strain and at 500 µg/plate in the TA 102 strain.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.