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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several genotoxicity tests were performed to evaluate the mutagen potential of DPTH.
Negative results were observed in the Ames test (OECD 471) and in the HPRT (OECD 476), however the MLA/TK test is positive.

Based on the in vitro data available, DPTH is considered to be not mutagenic.

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
Study period:
17 August 2011 - 01 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Target gene:
Histidine operon
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.
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.
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.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2011 - 08 November 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
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:
Experiments without S9 mix (3-hour treatment)
Using a treatment volume of 80 µL/20 mL, the selected dose-levels were as follows:
- 0.625, 1.25, 2.5, 5, 7.5 and 10 µg/mL for the first experiment,
- 0.156, 0.313, 0.625, 1.25, 1.88, 2.5 and 5 µg/mL for the second experiment.

Experiments with S9 mix (3-hour treatment)
Using a treatment volume of 80 µL/20 mL, the selected dose-levels were as follows:
- 1.25, 2.5, 5, 10, 20 and 30 µg/mL for the first experiment,
- 1.25, 2.5, 5, 10, 15, 20 and 30 µg/mL for 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: culture medium, water for injections, dimethylsulfoxide (DMSO), ethanol, acetone and tetrahydrofuran (THF). A suspension in DMSO, which was found homogeneous to the naked eye, was therefore selected for the treatments.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cultures of 20 mL at 5 x 105 cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO2 humidified incubator.

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
Evaluation criteria:
Positive result defined as:
- At least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 E-6
- A dose-related trend is demonstrated by a statistically significant trend test
- Unless clearly positive, the reproducibility should be confirmed

Negative results defined as:
- No evidence of mutagenicity at concentrations inducing moderate cytotoxicity (10% < Adj. RTG <20%), or
- If there no culture has 10% < Adj. RTG <20%:
¿ at least one negative data point with 20% < Adj. RTG <25% + negative data from 20% < Adj. RTG <100%, or
¿ at least one negative data point with 1% < Adj. RTG <10% + negative data from 25% < Adj. RTG <100%
Statistics:
This statistical analysis focuses on the slope of the regression line: If there is a significant linear relationship between the mutation frequency and the dose, the slope is not equal to zero.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
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 (Tables 1 and 2)
In the solubility assay, the test item was found indissoluble in the vehicles usually used for this type of assay: culture medium, water for injections, dimethylsulfoxide (DMSO), ethanol, acetone and tetrahydrofuran (THF). A suspension in DMSO, which was found homogeneous to the naked eye, was therefore selected for the treatments.
Using a treatment volume of 0.4%, the dose-levels selected for the treatment of the first preliminary test were 0.823, 2.47, 7.41, 22.2, 66.7 and 200 µg/mL.
At 200 µg/mL, the pH was approximately 7.4 (as for the vehicle control) and the osmolality was equal to 380 mOsm/kg H2O (384 mOsm/kg H2O for the vehicle control).
A precipitate was observed in the culture medium at dose-levels = 22.2 µg/mL and = 66.7 µg/mL at the end of the 3- and 24-hour treatment periods, respectively.
Following the 3-hour treatment without S9 mix, a moderate to severe toxicity was induced at all dose-levels, as shown by a 53-100% decrease in the Adjusted Relative Total Growth (Adj. RTG).
Following the 3-hour treatment with S9 mix, a slight to severe toxicity was induced at dose-levels = 7.41 µg/mL, as shown by a 38-100% decrease in the Adj. RTG.
Following the 24-hour treatment without S9 mix, a severe toxicity was induced at all dose-levels, as shown by a 100% decrease in the Adj. RTG. Due to the high level of cytotoxicity induced in the first preliminary test, a complementary preliminary test was undertaken with the same conditions but using a lower range of dose-levels as follows: 0.031, 0.063, 0.125, 0.25, 0.5 and 1 µg/mL. A moderate to severe toxicity was induced at dose-levels = 0.063 µg/mL, as shown by a 46-100% decrease in the Adj. RTG.


MUTAGENICITY EXPERIMENTS (Tables 3 to 10)
The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

Since the test item was found toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).

Experiments without S9 mix (3-hour treatment)
Cytotoxicity : In the first experiment, a marked to severe toxicity was induced at all dose-levels, as shown by a 75-99% decrease in the Adj. RTG.
In the second experiment, a slight to marked toxicity was induced at all dose levels, as shown by a 33-78% decrease in the Adj. RTG.
Mutagenicity : In the first experiment, a noteworthy increase in the mutation frequency was induced at 2.5 µg/mL, which showed a 88% decrease in the Adj. RTG. At higher dose-levels, a severe toxicity was noted (93-99% decrease in the Adj. RTG), preventing the interpretation of the corresponding mutation frequencies. The increase exceeded the Global Evaluation Factor (GEF) of 126 x 10-6 and a dose-response relationship was statistically demonstrated. Therefore, this result met the criteria for a positive response.
In the second experiment, a noteworthy increase in the mutation frequency was observed at 5 µg/mL, which showed a 78% decrease in the Adj. RTG. This increase exceeded the GEF and a dose-response relationship was statistically demonstrated. Therefore, this result met the criteria for a positive response. Compared to the vehicle control, the mutation frequencies were increased of up to 68 x 10-6 and 138 x 10-6, for the large and small colonies respectively (tables 4 and 6). This might indicate that the test item induced chromosome damages as well as point mutations.

Experiments with S9 mix (3-hour treatment)
A precipitate was observed in the culture medium at dose-levels = 20 µg/mL at the end of the 3 hour treatment periods.
Cytotoxicity: A slight to severe toxicity was induced at dose-levels = 5 µg/mL, as shown by a 42-96% (first experiment) and 34-89% (second experiment) decrease in the Adj. RTG.
Mutagenicity: In the first experiment, a noteworthy increase in the mutation frequency was induced at 20 µg/mL, which showed a 88% decrease in the adj. RTG. At 30 µg/mL, a severe toxicity (96% decrease in Adj. RTG) prevented the interpretation of the results. The increase exceeded the GEF and a dose-response relationship was statistically demonstrated. Therefore, this result met the criteria for a positive response.
In the second experiment, a noteworthy increase in the mutation frequency was induced at dose level = 15 µg/mL, which showed up to 89% decrease in the adj. RTG. The increase exceeded the GEF at 15 and 30 µg/mL and a dose-response relationship was statistically demonstrated. Therefore, this result met the criteria for a positive response.
Compared to the vehicle control, the mutation frequencies were increased of up to 92 x 10-6 and 149 x 10-6, for the large and small colonies respectively (tables 8 and 10). This might indicate that the test item induced chromosome damages as well as point mutations.

Conclusions:
The test item showed mutagenic activity in the mouse lymphoma assay, both in the presence and in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells.

The study was performed according to international guidelines (OECD 476 and Council Regulation (EC) No. 440/2008 of 30 May 2008, Part B17) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After preliminary toxicity tests, Dipentamethylenethiuram hexasulfide, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO2humidified incubator.

 

Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

The test item was suspended in dimethylsulfoxide (DMSO).

 

The dose-levels for the positive controls were as follows:

.           without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL,

.           with S9 mix: cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

 

Results

The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

 

Since the test item was found toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).

 

Experiments without S9 mix (3-hour treatment)

Using a treatment volume of 80 µL/20 mL, the selected dose-levels were as follows:

.           0.625, 1.25, 2.5, 5, 7.5 and 10 µg/mL for the first experiment,

.           0.156, 0.313, 0.625, 1.25, 1.88, 2.5 and 5 µg/mL for the second experiment.

 

Cytotoxicity

In the first experiment, a marked to severe toxicity was induced at all dose-levels, as shown by a 75-99% decrease in the Adj. RTG.

In the second experiment, a slight to marked toxicity was induced at all dose-levels, as shown by a 33-78% decrease in the Adj. RTG.

 

Mutagenicity

Noteworthy increases in the mutation frequency, exceeding the Global Evaluation Factor (GEF) of 126 x 10-6 were noted in both experiments, together with a statistically significant dose-response relationship. Therefore, these results met the criteria for a positive response.

 

Experiments with S9 mix (3-hour treatment)

Using a treatment volume of 80 µL/20 mL, the selected dose-levels were as follows:

.           1.25, 2.5, 5, 10, 20 and 30 µg/mL for the first experiment,

.           1.25, 2.5, 5, 10, 15, 20 and 30 µg/mL for the second experiment.

 

A precipitate was observed in the culture medium at dose-levels = 20 µg/mL at the end of the 3-hour treatment periods.

 

Cytotoxicity

A slight to severe toxicity was induced at dose-levels = 5 µg/mL, as shown by a 42-96% (first experiment) and 34-89% (second experiment) decrease in the Adj. RTG.

 

Mutagenicity

Noteworthy increases in the mutation frequency, exceeding the Global Evaluation Factor (GEF) of 126 x 10-6 were noted in both experiments, together with a statistically significant dose-response relationship. Therefore, these results met the criteria for a positive response.

 

Conclusion

The test item showed mutagenic activity in the mouse lymphoma assay, both in the presence and in the absence ofliver metabolizing system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
HPRT
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August -November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells (HPRT)
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Concentrations selected for the Mutation Experiment were based on the results of the cytotoxicity Range-Finder Experiment.

Range finder (+/-S9): six concentrations were tested in the absence and presence of S-9, ranging from 3.125 to 100 µg/mL (limited by solubility in culture medium and allowing for 1% v/v additions of the formulated test article to the test system).
First mutation experiment : eleven concentrations, ranging from 0.5 to 16 µg/mL in the absence of S-9 and from 2 to 30 µg/mL in the presence of S-9, were tested.
Second Mutation experiment : ten concentrations, ranging from 0.25 to 15 µg/mL in the absence of S-9 and from 0.5 to 20 µg/mL in the presence of S-9, were tested.
Vehicle / solvent:
dimethyl formamide (DMF)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
At least 10^7 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article, or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.

DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control


- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.
Rationale for test conditions:
Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 3.125 to 100 µg/mL (limited by solubility in culture medium and allowing for 1% v/v additions of the formulated test article to the test system). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (25 to 100 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration tested in the absence and presence of S-9
(100 µg/mL). The highest concentrations to give >10% RS were 6.25 µg/mL in the absence of S-9 and 12.5 µg/mL in the presence of S-9, which gave 22% and 18% RS, respectively.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (100 µg/mL), compared to the concurrent vehicle controls.

In Experiment 1, eleven concentrations, ranging from 0.5 to 16 µg/mL in the absence of S-9 and from 2 to 30 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest five concentrations tested in the absence of S-9 (8 to 16 µg/mL) and in the presence of S-9 (15 to 30 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected for analysis of viability and 6TG resistance. The highest concentrations analysed were 7 µg/mL in the absence of S-9 and 12 µg/mL in the presence of S-9, which gave 11% and 12% RS, respectively.

In Experiment 2, ten concentrations, ranging from 0.25 to 15 µg/mL in the absence of S-9 and from 0.5 to 20 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest two concentrations tested in the absence of S-9 (10 and 15 µg/mL) and the highest three concentrations tested in the presence of S-9 (12 to 20 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected for analysis of viability and 6TG resistance. The highest concentrations analysed were
8 µg/mL in the absence of S-9 and 10 µg/mL in the presence of S-9, which gave 11% and 28% RS, respectively. The % RS value observed at 10 µg/mL in the presence of S-9 was sufficiently close to 20% RS to be considered acceptable.

Genotoxicity
When tested up to toxic concentrations in the absence of S-9 in Experiment 1, statistically significant increases in mean MF, compared to the mean vehicle control MF values, were observed at the lowest two concentrations analysed (0.5 and 1 µg/mL) but no statistically significant linear trend was observed over the concentration range analysed. The mean MF values at 0.5 and 1 µg/mL were 5.56 and 5.64 mutants per 106 viable cells, respectively, both of which were within the current historical vehicle control range (0.51 to 5.90 mutants per 106 viable cells) based on the last 20 experiments performed in this laboratory, prior to Experiment 1 of this study. As all mean MF values were within the historical vehicle control range and there was no statistically significant linear trend, the statistically significant increases in mean MF at the lowest two concentrations analysed were considered not biologically relevant.
When tested up to toxic concentrations in the presence of S-9, a statistically significant increase in mean MF, compared to the mean vehicle control MF values, was observed at the second highest concentration analysed (10 µg/mL, giving 11% RS, which was at the upper limit of cytotoxicity for this type of study) and a statistically significant linear trend (p=0.01) was observed. However, the mean MF value at 10 µg/mL was 5.13 mutants per 106 viable cells, which was within the current historical vehicle control range based on the last 20 experiments performed in this laboratory, prior to Experiment 1 of this study (0.77 to 6.11 mutants per 106 viable cells). Furthermore, although the linear trend test was statistically significant, the mean MF at the highest concentration analysed (12 µg/mL, giving 12% RS) was 2.60 mutants per 106 viable cells, which was well within the historical vehicle control range. As all mean MF values were within the historical vehicle control range and
there was no clear concentration-related response, the statistically significant increases in mean MF at a single concentration analysed was considered not biologically relevant.
Based on the results of Experiment 1, a confirmatory experiment (designated Experiment 2) was performed in the absence and presence of S-9 in order to investigate the sporadic increases in MF observed under both treatment conditions. When tested up to toxic concentrations in the absence and presence of S-9 in Experiment 2, no statistically significant increases in mean MF, compared to the mean vehicle control MF values, were observed at any concentration analysed and there were no statistically significant linear trends.
Overall, the sporadic, statistically significant increases in mean MF at the lowest two concentrations (0.5 and 1 µg/mL) analysed in the absence of S-9 in Experiment 1, were small in magnitude (5.56 and 5.64 mutants per 106 viable cells, respectively, compared to the mean vehicle control MF of 1.49 mutants per 106 viable cells). Furthermore, there were no statistically significant increases in mean MF in the absence of S-9 in Experiment 2 at 0.5 and 1 µg/mL (3.48 and 5.47 mutants per 106 viable cells, respectively, compared to the mean vehicle control MF of 6.27 mutants per 106 viable cells) or at any other concentration analysed. Similarly, the statistically significant increase in mean MF at 10 µg/mL (giving 11% RS) in the presence of S-9 in Experiment 1 (5.13 mutants per 106 viable cells, compared to the mean vehicle control MF of 1.69 mutants per 106 viable cells) was not reproducible at the same concentration (giving 28% RS) in the presence of S-9 in Experiment 2
(1.84 mutants per 106 viable cells, compared to the mean vehicle control MF of 2.19 mutants per 106 viable cells). The statistically significant increases in the absence and presence of S-9 in Experiment 1 were therefore considered not biologically relevant.
Conclusions:
It is concluded that Dipentamethylenethiuram hexasulfide did not induce biologically relevant increases in mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) in two independent experiments under the experimental conditions described.
Executive summary:

Dipentamethylenethiuram hexasulfide was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two Mutation Experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in dimethyl formamide (DMF). A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 3.125 to 100 µg/mL (limited by solubility in culture medium and allowing for 1% v/v additions of the formulated test article to the test system). The highest concentrations to give >10% relative survival (RS) were 6.25 µg/mL in the absence of S-9 and 12.5 µg/mL in the presence of S-9, which gave 22% and 18% RS, respectively.

In Experiment 1, eleven concentrations, ranging from 0.5 to 16 µg/mL in the absence of S-9 and from 2 to 30 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 7 µg/mL in the absence of S-9 and 12 µg/mL in the presence of S-9, which gave 11% and 12% RS, respectively.

In Experiment 2, ten concentrations, ranging from 0.25 to 15 µg/mL in the absence of S-9 and from 0.5 to 20 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG were 8 µg/mL in the absence of S-9 and 10 µg/mL in the presence of S-9, which gave 11% and 28% RS, respectively. The % RS value observed at 10 µg/mL in the presence of S-9 was sufficiently close to 20% RS to be considered acceptable.

Vehicle and positive control treatments were included in the Mutation Experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within (or very close to) acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) without S-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore the study was accepted as valid.

When tested up to toxic concentrations in the absence of S-9 in Experiment 1, statistically significant increases in mean MF, compared to the mean vehicle control MF values, were observed at the lowest two concentrations analysed (0.5 and 1 µg/mL) but no statistically significant linear trend was observed over the

concentration range analysed. The mean MF values at 0.5 and 1 µg/mL were 5.56 and 5.64 mutants per 106 viable cells, respectively, both of which were within the current historical vehicle control range (0.51 to 5.90 mutants per 106 viable cells) based on the last 20 experiments performed in this laboratory, prior to Experiment 1 of this study. As all mean MF values were within the historical vehicle control range and there was no statistically significant linear trend, the statistically significant increases biologically relevant.

When tested up to toxic concentrations in the presence of S-9, a statistically significant increase in mean MF, compared to the mean vehicle control MF values,

was observed at the second highest concentration analysed (10 µg/mL, giving 11% RS, which was at the upper limit of cytotoxicity for this type of study) and a

statistically significant linear trend (p=0.01) was observed. However, the mean MF value at 10 µg/mL was 5.13 mutants per 106 viable cells, which was within the current historical vehicle control range based on the last 20 experiments performed in this laboratory, prior to Experiment 1 of this study (0.77 to 6.11 mutants per 106 viable cells). Furthermore, although the linear trend test was statistically significant, the mean MF at the highest concentration analysed (12 µg/mL, giving 12% RS) was 2.60 mutants per 106 viable cells, which was well within the historical vehicle control range. As all mean MF values were within the historical vehicle control range and there was no clear concentration-related response, the statistically significant increase in mean MF at a single concentration analysed was considered not biologically relevant.

Based on the results of Experiment 1, a confirmatory experiment (designed Experiment 2) was performed in the absence and presence of S-9 in order to

investigate the sporadic increases in MF observed under both treatment conditions. When tested up to toxic concentrations in the absence and presence of S-9 in

Experiment 2, no statistically significant increases in mean MF, compared to the mean vehicle control MF values, were observed at any concentration analysed and there were no statistically significant linear trends.

Overall, the sporadic, statistically significant increases in mean MF at the lowest two concentrations (0.5 and 1 µg/mL) analysed in the absence of S-9 in Experiment 1, were small in magnitude (5.56 and 5.64 mutants per 106 viable cells, respectively, compared to the mean vehicle control MF of 1.49 mutants per 106 viable cells). Furthermore, there were no statistically significant increases in mean MF in the absence of S-9 in Experiment 2 at 0.5 and 1 µg/mL (3.48 and 5.47 mutants per 106 viable cells, respectively, compared to the mean vehicle control MF of 6.27 mutants per 106 viable cells) or at any other concentration analysed. Similarly, the statistically significant increase in mean MF at 10 µg/mL (giving 11% RS) in the presence of S-9 in Experiment 1 (5.13 mutants per 106 viable cells, compared to the mean vehicle control MF of 1.69 mutants per 106 viable cells) was not reproducible at the same concentration (giving 28% RS) in the presence of S-9 in Experiment 2 (1.84 mutants per 106 viable cells, compared to the mean vehicle control MF of 2.19 mutants per 106 viable cells). The statistically significant increases in the absence and presence of S-9 in Experiment 1 were therefore considered not biologically relevant.

It is concluded that Dipentamethylenethiuram hexasulfide did not induce biologically relevant increases in mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) in two independent experiments under the experimental conditions described.

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

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test (OECD 474) was performed on DPTH and shows negative results.

Based on the in vivo study, DPTH is considered to be not clastogen.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March 2012 - 19 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 6 weeks old on the day of treatment
- Mean body weight at study initiation: the mean body weight was 181 g for males (ranging from 170 g to 200 g) and 135 g for females (ranging from 122 g to 147 g)
- Fasting period before study: no
- Housing: the animals were housed by three to five, by sex and group, in polycarbonate cages with stainless lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 March 2012 to 19 April 2012.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: the vehicle was 0.5% aqueous methylcellulose solution prepared using purified water, obtained by reverse osmosis using a ELIX 5 plus apparatus and methylcellulose

- Amount of vehicle (if gavage or dermal): 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All the concentrations and dose-levels of the test item were expressed as active item, taking into account the purity of the test item. A correction factor of 1.073 was used.
The test item was ground to a fine powder using a mortar and pestle and suspended in the vehicle.
The preparations were then homogenized using a magnetic stirrer and maintained under agitation throughout the treatment period. Dosage forms were prepared within the 4 hours before use, and then kept at room temperature and protected from light until use. They were delivered to the study room in brown flasks, at room temperature.
For the main test, the target dose-levels were 500, 1000, 1500 mg/kg/day (males only) and 2000 mg/kg/day (females only). Thus, using a treatment volume of 20 mL/kg, four dose formulations were prepared the concentrations of 25, 50, 75 and 100 mg/mL.
Duration of treatment / exposure:
Two treatments separated by 24 hours.
Frequency of treatment:
One treatment per day.
Post exposure period:
Sacrifice: 24 hours after the last treatment.
Remarks:
Doses / Concentrations:
500, 1000, 1500 (males only) and 2000 mg/kg/day (females only)
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females at 500 and 1000 mg/kg/day.
8 males at 1500 and 8 females and 2000 mg/kg/day.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 10 mL/kg.
Tissues and cell types examined:
Bone marrow: polychromatic (PE) and normochromatic (NE) erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In order to determine the highest dose-level for use in the cytogenetic study, several preliminary tests were performed on groups of six animals (three males and three females). Clinical signs and any mortality were recorded for a period of 48 hours following the first treatment. At the end of this period, the animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital, and then killed by cervical dislocation.

In the absence of any relevant information on the test item toxicity by intraperitoneal route and in agreement with the Sponsor, the starting dose-level was 1000 mg/kg/day (group 1) at a dose volume of 10 mL/kg.

The other dose-levels tested (groups 2 and 3) were 1500 and 2000 mg/kg/day, respectively, at a dose-volume of 20 mL/kg.

Clinical signs and any mortality were recorded over a period of 48 hours following the first treatment. At the end of this period, the animals were deeply anesthetized by an intraperitoneal injection of pentobarbital sodium, then killed by cervical dislocation.

SAMPLING TIMES:
At sacrifice, 24 h after the last treatment.

DETAILS OF SLIDE PREPARATION:
After sacrifice, the femurs were removed and bone marrow was flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the
supernatant was removed and the cells in the sediment were resuspended. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa and then coded for "blind" scoring.

METHOD OF ANALYSIS:
For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes; the
Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, there must be: a statistically significant increase in the frequency of MPE when compared to the vehicle control group. Reference to historical data or other considerations of biological relevance may be taken into account in the evaluation of data obtained.
Statistics:
no
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 to 2000 mg/kg (2 times)
- Clinical signs of toxicity in test animals: In order to select the top dose-level for the cytogenetic study, the dose-level of 1000 mg/kg/day was first administered twice, to three males and three females (group 1). The interval between each administration was 24 hours. No unscheduled mortality occurred as this dose-level. Both males and females showed hunched posture, ventral recumbency, staggering gait, hypoactivity, piloerection and/or thin appearance.
At the dose-level of 1500 mg/kg/day (group 2), no unscheduled mortality occurred. Male rats showed chromorhynorrhea, hunched posture, hypotonia, piloerection and/or thin appearance, while no clinical signs were observed in females.
At the dose-level of 2000 mg/kg/day (group 3), one out of the three males was found dead on day 3. Hypotonia and dyspnea were noted prior to its death. No mortality occurred in females. Clinical signs such as chromorhynorrhea, hunched posture, hypotonia, piloerection, dyspnea and/or thin appearance were noted in males, while females showed no clinical signs.

MORTALITY, CLINICAL SIGNS AND BODYWEIGHT (MAIN STUDY)
Male No. X29316 given 500 mg/kg/day was found dead on day 3. Prior to death, hunched posture and hypotonia were seen. Male No. X29327 given 1500 mg/kg/day was found dead on day 3. Prior to death, hunched posture, hypotonia, liquid feces and soiled anus were seen. Male No. X29328 given 1500 mg/kg/day was found dead on day 2. Prior to death, hunched posture and hypotonia were seen. No mortality occurred in females at any dose-levels.
Several clinical signs (Hunched posture, Thin appearance, Hypotonia) were observed in male and female rats at all tested doses. No clinical signs were observed in animals given the positive control (i.e. 15 mg/kg of CPA) or negative control.
Males given 500, 1000 and 1500 mg/kg/day of the test item, showed a mean body weight loss of 14, 15 and 15 g, respectively, between days 1 and 3 (vs. a mean body weight gain of 11 g in males given the vehicle).
Females given 500, 1000 and 2000 mg/kg/day of the test item, showed a mean body weight loss of 6, 3 and 8 g, respectively, over the same period (vs. a mean body weight gain of 10 g in females given the vehicle).

CYTOGENETIC TEST RESULTS (MAIN STUDY)
Cyclophosphamide induced statistically significant increases in the frequency of MPE (p < 0.01 in males and p < 0.05 in females), indicating the sensitivity of the test system under our experimental conditions. Thus the study was considered to be valid.
The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.
The mean values of MPE in the groups treated with the three dose-levels of test item (0.6, 0.8, 1.1 MPE/1000 PE for the male groups; 1.0, 1.9, 1.4 MPE/1000 PE for the female groups) were similar to those of their respective vehicle control animals (0.9 and 0.9 MPE/1000 PE for the males and females, respectively). No statistically significant differences were noted. Therefore, the criteria for a positive response were not met.
The mean values of PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group.

The following clinical signs were observed in surviving animals (main study):

 

Sex

Males

Females

Dose-levels (mg/kg/day)

0

500

1000

1500

0

500

1000

2000

Hunched posture

 

4

5

6

 

3

4

8

Thin appearance

 

3

5

5

 

1

4

6

Hypotonia

 

3

3

5

 

2

4

4

Chromorhynorrhea

 

 

1

 

 

 

 

 

Chromodacryorrhea

 

 

1

2

 

 

 

1

Dyspnea

 

 

1

 

 

 

 

 

Total affected animals

0/5

4/4

5/5

6/6

0/5

3/5

4/5

8/8

 

Conclusions:
The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats.

 

The study was performed according to the international guidelines (OECD guideline No. 474 and Council Regulation No. 440/2008 of 30 May 2008, Annex, Part B.12) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Sprague-Dawley rats received two intraperitoneal treatments of the test item at dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females, at a 24-hour interval. For the high-dose group only, three supplementary males and three supplementary females were also treated with the test item in case of mortality.

One group of five males and five females received the vehicle (0.5% methylcellulose) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.

 

The animals of the treated and vehicle control groups were sacrificed 24 hours after the last treatment and the animals of the positive control group were sacrificed 24 hours after the single treatment. Bone marrow smears were then prepared.

 

For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).

 

Results

In accordance with the criteria specified in the international guideline and on the basis of the results of the preliminary test, the highest dose-level selected for the males was 1500 mg/kg/day, since a higher dose-level was expected to induce mortality; and the highest dose-level selected for the females was 2000 mg/kg/day, this dose being the highest recommended one.

 

Two out of the eight males given 1500 mg/kg/day were found dead on day 2 or 3 and one male given 500 mg/kg/day was found dead on day 3.

No mortality occurred in females at any dose-levels.

 

Several clinical signs (Hunched posture, Thin appearance, Hypotonia) were observed in male and female rats at all tested doses. No clinical signs were observed in animals given the positive control (i.e. 15 mg/kg of CPA) or negative control.

 

Cyclophosphamide induced statistically significant increases in the frequency of MPE (p < 0.01 males and p < 0.05 females), indicating the sensitivity of the test system under our experimental conditions. Thus the study was considered to be valid.

 

The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.

 

The mean values of MPE in the groups treated with the three dose-levels of test item (0.6, 0.8, 1.1 MPE/1000 PE for the male groups; 1.0, 1.9, 1.4 MPE/1000 PE for the female groups) were similar to those of their respective vehicle control animals (0.9 and 0.9 MPE/1000 PE for the males and females, respectively).No statistically significant differences were noted.Therefore, the criteria for a positive response were not met.

 

The mean values of PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group.

 

Conclusion

The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females.

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

Additional information

Bacterial reverse mutation assay / Ames test (OECD 471):

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium (OECD 471).

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. The test item was suspended in dimethylsulfoxide (DMSO).

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.

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

In vitro mammalian cell gene mutation assay / MLA/TK assay (OECD 476):

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells (OECD 476). After preliminary toxicity tests, Dipentamethylenethiuram hexasulfide, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. The test item was suspended in dimethylsulfoxide (DMSO). The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid. Since the test item was found toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).

In the first experiment without S9 mix (3 -hour treatment), a marked to severe toxicity was induced at all dose-levels, as shown by a 75-99% decrease in the Adj. RTG. In the second experiment without S9, a slight to marked toxicity was induced at all dose-levels, as shown by a 33-78% decrease in the Adj. RTG. Noteworthy increases in the mutation frequency, exceeding the Global Evaluation Factor (GEF) of 126 x 10^-6 were noted in both experiments without S9 mix, together with a statistically significant dose-response relationship. Therefore, these results met the criteria for a positive response.

In the experiments with S9 mix (3-hour treatment) : A precipitate was observed in the culture medium at dose-levels = 20 µg/mL at the end of the 3-hour treatment periods. A slight to severe toxicity was induced at dose-levels = 5 µg/mL, as shown by a 42-96% (first experiment) and 34-89% (second experiment) decrease in the Adj. RTG. Noteworthy increases in the mutation frequency, exceeding the Global Evaluation Factor (GEF) of 126 x 10^-6 were noted in both experiments with S9 mix, together with a statistically significant dose-response relationship. Therefore, these results met the criteria for a positive response.

The test item showed mutagenic activity in the mouse lymphoma assay, both in the presence and in the absence of liver metabolizing system.

In vitro mammalian cell gene mutation assay / HPRT assay (OECD 476):

Dipentamethylenethiuram hexasulfide was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two Mutation Experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in dimethyl formamide (DMF). A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 3.125 to 100 µg/mL (limited by solubility in culture medium and allowing for 1% v/v additions of the formulated test article to the test system). The highest concentrations to give >10% relative survival (RS) were 6.25 µg/mL in the absence of S-9 and 12.5 µg/mL in the presence of S-9, which gave 22% and 18% RS, respectively.

In Experiment 1, eleven concentrations, ranging from 0.5 to 16 µg/mL in the absence of S-9 and from 2 to 30 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 7 µg/mL in the absence of S-9 and 12 µg/mL in the presence of S-9, which gave 11% and 12% RS, respectively.

In Experiment 2, ten concentrations, ranging from 0.25 to 15 µg/mL in the absence of S-9 and from 0.5 to 20 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG were 8 µg/mL in the absence of S-9 and 10 µg/mL in the presence of S-9, which gave 11% and 28% RS, respectively. The % RS value observed at 10 µg/mL in the presence of S-9 was sufficiently close to 20% RS to be considered acceptable.

Vehicle and positive control treatments were included in the Mutation Experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within (or very close to) acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) without S-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore the study was accepted as valid.

When tested up to toxic concentrations in the absence of S-9 in Experiment 1, statistically significant increases in mean MF, compared to the mean vehicle control MF values, were observed at the lowest two concentrations analysed (0.5 and 1 µg/mL) but no statistically significant linear trend was observed over the

concentration range analysed. The mean MF values at 0.5 and 1 µg/mL were 5.56 and 5.64 mutants per 106 viable cells, respectively, both of which were within the current historical vehicle control range (0.51 to 5.90 mutants per 106 viable cells) based on the last 20 experiments performed in this laboratory, prior to Experiment 1 of this study. As all mean MF values were within the historical vehicle control range and there was no statistically significant linear trend, the statistically significant increases biologically relevant.

When tested up to toxic concentrations in the presence of S-9, a statistically significant increase in mean MF, compared to the mean vehicle control MF values,

was observed at the second highest concentration analysed (10 µg/mL, giving 11% RS, which was at the upper limit of cytotoxicity for this type of study) and a

statistically significant linear trend (p=0.01) was observed. However, the mean MF value at 10 µg/mL was 5.13 mutants per 106 viable cells, which was within the current historical vehicle control range based on the last 20 experiments performed in this laboratory, prior to Experiment 1 of this study (0.77 to 6.11 mutants per 106 viable cells). Furthermore, although the linear trend test was statistically significant, the mean MF at the highest concentration analysed (12 µg/mL, giving 12% RS) was 2.60 mutants per 106 viable cells, which was well within the historical vehicle control range. As all mean MF values were within the historical vehicle control range and there was no clear concentration-related response, the statistically significant increase in mean MF at a single concentration analysed was considered not biologically relevant.

Based on the results of Experiment 1, a confirmatory experiment (designed Experiment 2) was performed in the absence and presence of S-9 in order to

investigate the sporadic increases in MF observed under both treatment conditions. When tested up to toxic concentrations in the absence and presence of S-9 in

Experiment 2, no statistically significant increases in mean MF, compared to the mean vehicle control MF values, were observed at any concentration analysed and there were no statistically significant linear trends.

Overall, the sporadic, statistically significant increases in mean MF at the lowest two concentrations (0.5 and 1 µg/mL) analysed in the absence of S-9 in Experiment 1, were small in magnitude (5.56 and 5.64 mutants per 106 viable cells, respectively, compared to the mean vehicle control MF of 1.49 mutants per 106 viable cells). Furthermore, there were no statistically significant increases in mean MF in the absence of S-9 in Experiment 2 at 0.5 and 1 µg/mL (3.48 and 5.47 mutants per 106 viable cells, respectively, compared to the mean vehicle control MF of 6.27 mutants per 106 viable cells) or at any other concentration analysed. Similarly, the statistically significant increase in mean MF at 10 µg/mL (giving 11% RS) in the presence of S-9 in Experiment 1 (5.13 mutants per 106 viable cells, compared to the mean vehicle control MF of 1.69 mutants per 106 viable cells) was not reproducible at the same concentration (giving 28% RS) in the presence of S-9 in Experiment 2 (1.84 mutants per 106 viable cells, compared to the mean vehicle control MF of 2.19 mutants per 106 viable cells). The statistically significant increases in the absence and presence of S-9 in Experiment 1 were therefore considered not biologically relevant.

It is concluded that Dipentamethylenethiuram hexasulfide did not induce biologically relevant increases in mutation at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) in two independent experiments under the experimental conditions described.

In vivo micronucleus test in rats (OECD 474):

The objective of this study was to evaluate the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats. In the main study, three groups of five male and five female Sprague-Dawley rats received two intraperitoneal treatments of the test item at dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females, at a 24-hour interval. The animals of the treated and vehicle control groups were sacrificed 24 hours after the last treatment and the animals of the positive control group were sacrificed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).

Two out of the eight males given 1500 mg/kg/day were found dead on day 2 or 3 and one male given 500 mg/kg/day was found dead on day 3.

No mortality occurred in females at any dose-levels. Several clinical signs (Hunched posture, Thin appearance, Hypotonia) were observed in male and female rats at all tested doses. No clinical signs were observed in animals given the positive control (i.e. 15 mg/kg of CPA) or negative control.

Cyclophosphamide induced statistically significant increases in the frequency of MPE (p < 0.01 males and p < 0.05 females), indicating the sensitivity of the test system under our experimental conditions. Thus the study was considered to be valid. The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.

The mean values of MPE in the groups treated with the three dose-levels of test item (0.6, 0.8, 1.1 MPE/1000 PE for the male groups; 1.0, 1.9, 1.4 MPE/1000 PE for the female groups) were similar to those of their respective vehicle control animals (0.9 and 0.9 MPE/1000 PE for the males and females, respectively).No statistically significant differences were noted.Therefore, the criteria for a positive response were not met.The mean values of PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group. The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 1500 mg/kg/day in males and 500, 1000 and 2000 mg/kg/day in females.

Justification for classification or non-classification

Based on the available data, no classification for mutagenicity is required for Dipentamethylenethiuram hexasulfide according to the Regulation EC n°1272/2008.

Even if positive results were obtained in the MLA/TK assay, negative results were obtained in the HPRT and AMES tests, suggesting that the DPTH did not induce gene mutations, and that the results of MLA/TK are false positive as it is the case for the most thiourea. Moreover, an in vivo micronucleus test on DPTH concluded that DPTH did not induce chromosome aberrations in rat. Based on the Weight-of-Evidence, Dipentamethylenethiuram hexasulfide is not genotoxic. Performing a new in vivo study is considered as not relevant according to the Annexe XI and for animal welfare.