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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2012-09-27 - 2013-02-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol (HiTEC 4313 Performance Additive)
- Substance type: organic
- Physical state: liquid
- Storage condition of test material: in the dark at ambient temperature

Method

Target gene:
thymidine kinase heterozygote system, where tk+tk- is mutated to tk-tk-
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The basic culture medium (R0P) was RPMI 1640 medium, supplemented with penicillin G (100 units/mL), streptomycin (100 μg/mL), sodium bicarbonate (1.125 g/L) and pluronic acid (0.05%, w/v).
For cell growth, heat-inactivated horse serum (10%, v/v) was added to R0P to give R10P.
The medium used during treatment for 4 h was R0P supplemented with 5% (v/v) heat-inactivated horse serum (R5P). The medium used during treatment for 24 h was R10P.
For colony formation, cloning medium was used, consisting of R0P supplemented with heat-inactivated horse serum (20%, v/v), sodium pyruvate (1.9 mM), and amphotericin B (fungizone) (2.5 μg/mL).
For selection of tk-tk- cells, cloning medium was supplemented with trifluorothymidine (TFT) at 3 μg/mL.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes, (The tk+tk- heterozygote cells grown in suspension spontaneously mutate to tk-tk- at a rate of 2 x 10-6 mutations/generation. These homozygous mutants were removed before testing began).

Other:
The cells grow in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium.
Some mutant cells divide at the normal rate, producing large colonies, while other cells divide at a distinctly slower rate, producing small colonies. A high proportion of large type colonies are associated with small chromosomal deletions or point mutations, while a large proportion of the small type colonies are associated with large chromosomal deletions. Assessment of the relative numbers of both colony types can provide information to support results obtained in bacterial mutation and chromosome aberration tests.
Test concentrations with justification for top dose:
0.1, 0.3, 1, 3.3, 10, 33.3, 100, 333.3 and 1000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [acetone]
- Justification for choice of solvent/vehicle: Information supplied by the Sponsor indicated that acetone was a suitable vehicle for use with
1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol. Acetone was therefore used to formulate the test item throughout the study.
- Other details: In this study, the vehicle control cultures were treated with acetone. In the toxicity tests, the concentration of acetone was 1 %, v/v, throughout. In the mutation experiments, the final concentration of acetone was reduced in the experiment in the absence of S9 mix with a 24 h exposure period to 0.5 %, v/v. This action was taken to improve cell growth following exposure, as it was noted that use of 1 % acetone, v/v resulted in reduced cell growth in the toxicity test.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Acetone 1 % v/v
Positive controls:
yes
Remarks:
In the absence of S9 mix (4 h exposure period) were: 250 μg/mL EMS, and 10 μg/mL MMS. In the presence of S9 mix 3-MC was used at concentrations of 2.5 and 10 μg/mL. EMS and 3-MC were dissolved in dimethylsulphoxide, while MMS was dissolved in water.
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: soft agar cloning method originally developed by Clive

DURATION
- Exposure duration: 4 / 24 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) at 3 μg/mL.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
Treatment (4 h Exposure Period)
On the day of the test (Day 0), samples of cell culture (in 5 mL R10P) were dispensed to sterile tubes containing R0P (3.9 mL). Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by the test formulation (0.1 mL). Vehicle control cultures received acetone (0.1 mL). Positive control cultures received the appropriate solution (0.1 mL). The final reaction mixture in all cultures contained 10 mL of cells, at a population density estimated at 6.0 x 10E5 cells/mL, in R5P medium.
All tubes were placed on a 10 r.p.m. rotating drum, inside an incubator set to maintain a temperature of 37°C, for 4 h. After this, the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated to give a cell density estimated at 3 x 10E5/mL.
The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10E5 cells/mL on day 1.

Extended Treatment (24 h Exposure)
An experiment is conducted using an extended 24 h exposure period, when the results of the first experiment in the absence of S9 mix are negative.The extended exposure period facilitates continuous exposure to the test item through > 1 cell cycle.
On the day of the test (day 0), samples of cell culture (in 10 mL R10P) were dispensed to sterile tubes containing R0P (7.9 mL). R50P (R0P:serum, 50:50, v/v) (2 mL) was added to each tube followed by the test formulation (0.1 mL). Vehicle control cultures received acetone (0.1 mL). Positive control cultures received the appropriate solution (0.1 mL). The final reaction mixture in all cultures contained 20 mL of cells, at a population density estimated at 3 x 10E5 cells/mL, in R10P medium.
(The larger volumes allow the same numbers of cells to be treated as in the experiments conducted at 4 h exposure, but at half the density. The lower density is required to allow cell growth during the exposure period. The serum concentration is not lowered, as some essential nutrients can become exhausted during the exposure period.)
All tubes were incubated on the rotating drum (as described above) for 24 h. After this (on day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and were then resuspended in R10P medium (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give an estimated 3 x 10E5 cells/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10E5 cells/mL on day 2.

Expression of Genetic Damage
On day 2 (4 h exposure) or day 3 (24 h exposure), cell counts were determined. The cell counts over the 2 or 3 days of the experiments provided a measure of suspension growth. This in turn provided a measure of RSG. This was used when choosing dose levels to carry through to final assessment, as no other measures of toxicity were known at the time the decision was required.
In this study, the treated cultures from the 5 highest concentrations of 1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol giving satisfactory cell survival were selected for final assessment in all 4 assays. The cultures were then assessed for expression of genetic damage. This was determined by performing parallel cloning assays for cloning efficiency and mutant selection. For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well.
For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 10E4 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well.
All dishes were placed in an incubator set to maintain a humid atmosphere of 5 % CO2: 95 % air (v/v) at 37 °C until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).
Evaluation criteria:
Criteria for a Positive Result: The global evaluation factor (GEF = mean of the global vehicle control distribution plus one standard deviation) of 126 mutants per million applies for this assay and biological significance startes at values > 126 mutants per million.
An experiment was considered positive:
• if one or more concentrations showed a biolog. signif. increase in mutant fraction and a significant linear trend.
• without a linear trend - if there was mitigating evidence. This might be the presence of a similar level of toxicity at all concentrations assessed. Then the confirmatory experiment should assess concentrations covering different levels of toxicity, to establish a linear trend.
A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition. Test items that gave a negative response in the standard exposure in the absence of S9 mix, but gave a positive response in the extended exposure, were liable to a confirmatory experiment with the extended exposure.
Criteria for a Negative Result: a test item was defined as non-mutagenic, provided data were obtained in both the absence and the presence of S9 mix that accompanied one or more of the following:
• the predetermined maximum concentration of 5000 μg/mL or 10 mM, whichever is lower
• the highest practicable concentration limited by the solubility or pH of the test item
• RTG in the range 10 - 20 %
It is acknowledged (Moore et al., 2002) that in some circumstances a chemical may be determined to be non-mutagenic when there was no treatment showing an RTG value of 10 - 20 %. These situations are as follows:
a) no evidence of mutagenic activity in a series of data points within 100 % to 20 % RTG and at least one data point between 20 % and 25 % RTG.
b) no evidence of mutagenic activity in a series of data points between 100 % to 25 % RTG and a data point between 10 % and 1 % RTG.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
All mean IMF values were well below the minimum 126 mutants per million required to indicate a biologically relevant increase, and therefore the test item was considered not to be demonstrating mutagenic activity.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at the 3 highest concentrations of 100, 333.3 and 1000 μg/mL in all 3 test conditions. This was of no consequence, as toxicity required the use of lower, nonprecipitating dose levels.

RANGE-FINDING/SCREENING STUDIES:
Toxicity Test: The exposure of the cells was similar to that described for the mutation assays with the exception that only one culture was prepared for each treatment.
The selection in the mutation experiments of the treatments for final assessment is dependent on suspension growth following treatment. The measure used to assess toxicity in the dose range-finding test was therefore relative suspension growth. The cell population densities were recorded over 2 days (following treatment) using a haemocytometer, then the total suspension growths were expressed as percentages of the vehicle control group (= relative suspension growth, or RSG).
The toxicity test was performed using the standard 4 h exposure period in the absence and presence of S9 mix. An additional toxicity test was performed in the absence of S9 mix with 24 h exposure to 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen Peroxide and Tert-nonanethiol, as a contingency against the later requirement for a full experiment using this extended exposure period.
Observations on the precipitation of 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol were made after dosing and at the end of the exposure period. Observations of pH change (colour change in indicator in RPMI medium) were made and if any change was noted, pH measurements were made.

The results of the toxicity tests showed that the substance was of a moderate to high level of toxicity. In the absence of S9 mix, a concentration of 3.3 μg/mL of test item after a 4 h treatment reduced cell suspension growth to 38.2 % of the vehicle control group, while a concentration of 10 μg/mL reduced suspension growth to 13.8 %. Concentrations of 33.3 μg/mL and higher were lethal.
However, it was rather less toxic in the presence of S9 mix. A concentration of 10 μg/mL of test item after a 4 h treatment reduced cell suspension growth to 49.2 %, while 33.3 μg/mL reduced suspension growth to 6.9 %. Concentrations of 100 μg/mL and higher were lethal.
When the exposure period in the absence of S9 mix was extended to 24 h, a concentration of 3.3 μg/mL of test item reduced suspension growth to 28.4 %. All higher concentrations were lethal.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Also in the experiment without S9 -mix (4 h), all values were well below the minimum of 126 mutants per million required to indicate a biologically relevant increase, with theexception of the 19.75 μg/mL group. This treatment gave an IMF value of 132 mutants per million: just above the threshold value. The relevance of this single increase was made questionable by the downward trend of the IMF at the higher, more toxic concentration of 27.25 μg/mL (74 mutants per million). Again, the marginally higher values for the treated groups resulted in the linear trend test showing significance (P < 0.001).

The RTG levels at the two highest assessed concentrations of 19.75 and 27.25 μg/mL were 19 % and 10 %, respectively, which are both definitive levels of toxicity. A call of inconclusive was made for the assay, and further assessment was deferred to the second experiment in the presence of S9 mix.

In the extended experiment, all mean IMF values were well below the minimum 126 mutants per million required to indicate a biologically relevant increase, and therefore the test item was considered not to be demonstrating mutagenic activity. Again, the IMF values for 1,3,4-Thiadiazolidine-2,5-

dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol were consistently marginally higher than the vehicle control group. This resulted in the test for linear trend in mutant fraction with concentration of test item showing significance (P < 0.001).

A call of not mutagenic was made for the assay.

In 2 fully acceptable assays in the absence of S9 mix (one with a 4 h exposure period and one with a 24 h exposure period), no 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol treatment resulted in a biologically relevant increase in mutant fraction. Both assays returned treatments showing a definitive level of toxicity. It is therefore concluded that the test item is not mutagenic in the absence of S9 mix.

In 2 fully acceptable assays in the presence of S9 mix, the test substance did not demonstrate a reproducible biologically relevant increase in mutant fraction. Both assays returned treatments showing a definitive level of toxicity. It is therefore concluded that the test item is not mutagenic in the presence of S9 mix.

It should be noted that small increases in the induced mutant fraction considered to be of no biological relevance were consistently obtained in the 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol treatment groups.

In conclusion, 1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix when tested in acetone at concentrations extending into the toxic range.

Table 1. Toxicity Test in the Absence of S9 Mix (4 h Exposure)
Chemical Concentration (µg/mL) Precipitation Suspension Count (x 105/mL) Total Suspension Growth Relative Suspension Growth %
Day1 Day 2
Acetone (100 µL added)   10.2 20.0 22.7 100.0
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 0.1   11.6 16.0 20.6 91.0
0.3   11.4 20.4 25.8 114.0
1.0   10.2 18.8 21.3 94.0
3.3   5.0 15.6 8.7 38.2
10.0   2.4 9.4 3.1 13.8
33.3   0.0 0.0 0.0 0.0
100.0 pptn 0.0 0.0 0.0 0.0
333.3 pptn 0.0 0.0 0.0 0.0
1000.0 pptn 0.0 0.0 0.0 0.0
pptn=Precipitation

Table 2. Toxicity Test in the Presence of S9 Mix (4 h Exposure)
Chemical Concentration (µg/mL) Precipitation Suspension Count (x 105/mL) Total Suspension Growth Relative Suspension Growth %
Day1 Day2
Acetone (100 µL added)   13.2 18.0 26.4 100.0
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 0.1   13.0 20.6 29.8 112.7
0.3   13.8 19.4 29.7 112.7
1.0   13.6 17.0 25.7 97.3
3.3   11.0 16.0 19.6 74.1
10.0   7.5 15.6 13.0 49.2
33.3   1.4 5.5 1.8 6.9
100.0 pptn 0.0 0.0 0.0 0.0
333.3 pptn 0.0 0.0 0.0 0.0
1000.0 pptn 0.0 0.0 0.0 0.0
pptn = Precipitation

Table 3. Toxicity Test in the Absence of S9 Mix (24 h Exposure)
Chemical Concentration (µg/mL) Precipitation Suspension Count (x 105/mL) Total Suspension Growth Relative Suspension Growth%
Day1 Day 2 Day3
Acetone (200 µL added)   13.0 8.7 15.7 65.8 100.0
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 0.1   10.6 8.3 17.4 56.7 86.2
0.3   9.0 9.2 17.0 52.1 79.3
1.0   8.4 7.6 15.4 36.4 55.4
3.3   4.6 8.0 13.7 18.7 28.4
10.0   0.2 0.0 0.0 0.0 0.0
33.3   0.0 0.0 0.0 0.0 0.0
100.0 pptn 0.0 0.0 0.0 0.0 0.0
333.3 pptn 0.0 0.0 0.0 0.0 0.0
1000.0 pptn 0.0 0.0 0.0 0.0 0.0
pptn = Precipitation

Table 4. Mutation Test in the Absence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 1)
Chemical Concentration (µg/mL) Relative Total Growth% Mutant Fraction(x10-6) IMF (Induced Mutant Fraction x 10-6) Ratio of Small to Large Colonies
Acetone (100 µL added) 100 91 N/A 3.51
EMS 250 74 893 802 0.62
MMS 10 40 1967 1876 2.32
1,3,4-Thiadiazolidine-2,5- 1.00 59 140 49 1.54
dithione, Reaction Products with Hydrogen Peroxide and 1.75 44 173 82 1.45
Tert-nonanethiol 3.25 26 128 37 1.19
  5.50 19 148 56 1.64
  8.50 16 175 84 1.61
  12.25 NPT NPT NPT NPT
  16.75 NPT NPT NPT NPT
  22.00 NPT NPT NPT NPT
IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group
N/A = Not Applicable
Test for linear trend of mutant fraction with concentration of 1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol=significant (P=0.001)
NPT = Not Plated - Toxic

Table 5. Mutation Test in the Presence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 2)
Chemical Concentration (µg/mL) Relative Total Growth % Mutant Fraction(x10-6) IMF (Induced Mutant Fraction x 10-6) Ratio of Small to Large Colonies
Acetone (100 µL added) 100 96 N/A 1.33
3-MC 2.5 53 1092 995 1.06
  10 35 1472 1375 1.68
1,3,4-Thiadiazolidine-2,5- 2.25 NPS NPS NPS NPS
dithione, Reaction Products with Hydrogen 4.75 55 134 38 1.04
Peroxide and Tert-nonanethiol 8.50 36 139 43 1.13
  13.50 24 157 61 1.07
  19.75 19 228 132 0.93
  27.25 10 171 74 1.49
  36.00 NPT NPT NPT NPT
  46.00 NPT NPT NPT NPT
IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group
N/A=Not Applicable
Test for linear trend of mutant fraction with concentration of 1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol=significant (P<0.001)
NPS = Not Plated - Surplus
NPT = Not Plated - Toxic

Table 6. Mutation Test in the Absence of S9 Mix (24 h Exposure) Summary of Means of Data (Assay 3)
Chemical Concentration(µg/mL) Relative Total Growth % Mutant Fraction(x10-6) IMF (Induced Mutant Fraction x 10-6) Ratio of Small to Large Colonies
Acetone (100 µL added) 100 130 N/A 0.90
EMS 100 82 1540 1410 0.49
MMS 5 43 2537 2407 1.37
1,3,4-Thiadiazolidine-2,5- 0.1 NPS NPS NPS NPS
dithione, Reaction Products with Hydrogen Peroxide and 0.4 NPS NPS NPS NPS
Tert-nonanethiol 1.0 66 204 74 0.80
  1.9 45 207 77 1.63
  3.1 35 200 71 1.07
  4.6 25 218 89 1.10
  6.4 14 214 84 0.99
  8.5 NPT NPT NPT NPT
IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group
N/A = Not Applicable
Test for linear trend of mutant fraction with concentration of 1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol = significant (P<0.001)
NPS = Not Plated - Surplus
NPT = Not Plated - Toxic

Table 7. Mutation Test in the Presence of S9 Mix (4 h Exposure) Summary of Means of Data (Assay 4)
Chemical Concentration(µg/mL) Relative Total Growth % Mutant Fraction (x 10-6) IMF (Induced Mutant Fraction x 10-6) Ratio of Small to Large Colonies
Acetone (100 µL added) 100 112 N/A 1.53
3-MC 2.5 45 1299 1187 0.91
  10 33 1448 1336 0.86
1,3,4-Thiadiazolidine-2,5- 2.25 NPS NPS NPS NPS
dithione, Reaction Products with Hydrogen 4.75 57 147 35 1.01
Peroxide and Tert-nonanethiol 8.50 29 205 94 0.79
  13.50 21 198 86 0.61
  19.75 13 216 104 0.77
  27.25 + (9) (343) (231) (0.76)
  36.00 NPT NPT NPT NPT
  46.00 NPT NPT NPT NPT
IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group
N/A = Not Applicable
Test for linear trend of mutant fraction with concentration of 1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol = significant (P<0.001)
NPS = Not Plated - Surplus
NPT = Not Plated - Toxic
+   = Toxic concentration resulting in unacceptable relative total growth (<10%). Data included for information, but excluded from assessment

Table 8. Historical Control Data
Mouse Lymphoma L5178Y Cell Historical Control Data from Recent Experiments (August 2006 to December 2011]
Vehicle Control Data
Vehicle Control S9 Exposure Time   Mutant Fraction x 10"6 No-effect Maximum (Induced Mutant Fraction) Mean Colony Size Ratio (Small/Large)
  Mean SD Range Mean SD Range
All, poolec 1 4h 59 SI 24 49-155 49 1.40 0.41 0.73-2.64
All, poolec 1 24h 50 89 32 47-194 60 1.36 0.50 0.60-2.94
All, poolec 1 + 4h 109 93 27 47-188 79 1.42 0.38 0.64-2.75
| — Each value is the mean of 4 replicate cultures
The No-effect Maximum represents the maximum difference recorded between the 2 pairs of vehicle control cultures in any experiment That is, the lower mean mutant fraction (x 10"6) is subtracted from the higher. This difference, when applied to the response from a mutagen, is termed the induced mutant fraction (IMF). Positive Control Data
Positive Control S9 Concentration (µg/mL) Exposure Time   Mutant Fraction x 10"6 RTG% Mean Colony Size Ratio (Small/Large)
  Mean SD Range Mean Range Mean SD Range
EMS - 250 4h 59 722 261 433-1560 59 38-79 0.54 0.11 0.29-0.84
MMS - 10 4h 59 1063 320 543-2094 38 22-75 2.22 0.58 1.39-3.79
EMS* - 150 24h 48 2226 833 1197-4475 33 8-92 0.35 0.10 0.14-0.63
EMS* - 100 24h 2 2882 - 2637-3126 31 24-38 0.41 - 0.32-0.49
MMS - 5 24 h 50 1938 460 1115-3204 33 17-91 1.65 0.44 0.86-2.94
3-MC + 2.5 4h 109 917 411 403-2277 60 14-96 1.27 0.35 0.67-3.61
3-MC + 10 4h 54 1086 458 515-2251 47 6-80 1.34 0.28 0.73-2.18
— Each value is the mean of 2 replicate cultures
EMS = Ethyl methanesulphonate
MMS = Methyl methanesulphonate
3-MC — 3-Methylcholanthrene
# After many years of use, 150 µg EMS/mL became too toxic in the 24 h test system, resulting in a recent reduction to 100 µg/mL.
Audited by: Alison McNaughton (Quality Assurance)
22 May 2012

Table 9.  Individual Suspension Growth Data Mutation Test in the Absence of S9 Mix (4 h Exposure) Suspension Growth (Assay 1)
Chemical Concentration (µg/rnL) Observations Suspension Count (x 105/mL) Total Suspension Growth Relative Suspension Growth (%)
Day 1 Day 2
Vehicle Control Mean _       19.6  
Acetone (100 µL added)   10.5 18.4 21.5 109.6
9.0 18.0 18.0 91.9
10.0 18.0 20.0 102.1
10.9 15.6 18.9 96.4
EMS 250 - 8.5 14.8 14.0 71.4
9.8 13.0 14.2 72.3
MMS 10 - 8.0 14.6 13.0 66.2
10.4 13.2 15.3 77.9
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 1.00 - 6.0 16.0 10.7 54.4
8.1 12.6 11.3 57.9
1.75 - 6.1 13.6 9.2 47.1
4.8 14.8 7.9 40.3
3.25 - 3.5 14.8 5.8 29.4
3.7 13.6 5.6 28.5
5.50 - 2.5 11.0 3.7 18.7
2.5 14.0 4.7 23.8
Suspension Growth (Assay 1 Continued)
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 8.50 - 1.8 7.5 2.5 12.8
2.7 7.0 2.3 11.9
12.25 - 0.5 2.0 0.7 3.4
0.8 3.0 1.0 5.1
16.75 - 0.2 0.2 0.1 0.3
0.2 0.2 0.1 0.3
22.00 - 0.8 0.0 0.0 0.0
1.0 0.0 0.0 0.0

Table 9 (continued). Mutation Test in the Presence of S9 Mix (4 h Exposure) Suspension Growth (Assay 2)
Chemical Concentration(µg/mL) Observations Suspension Count (x 105/mL) Total Suspension Growth Relative Suspension Growth (%)
Day 1 Day 2
Vehicle Control Mean _       23.9  
Acetone (100 µL added)   12.2 17.2 23.3 97.6
11.0 19.6 24.0 100.3
10.9 18.0 21.8 91.3
14.0 17.0 26.4 110.7
3-MC 2.5 - 7.6 17.0 14.4 60.1
8.4 16.4 15.3 64.1
  10 - 7.1 12.0 9.5 39.6
8.1 13.0 11.7 49.0
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 2.25 - 9.5 14.8 15.6 65.4
9.9 17.6 19.4 81.1
4.75 - 6.7 16.0 11.9 49.9
8.2 13.0 11.8 49.6
8.50 - 4.4 14.0 6.8 28.7
4.4 16.0 7.8 32.8
13.50 - 2.5 14.8 4.9 20.7
2.8 15.6 5.2 21.8
Suspension Growth (Assay 2 Continued)
1,3,4-Thiadiazolidine-2,5-dithione, Reaction Products with Hydrogen Peroxide and Tert-nonanethiol 19.75 - 1.1 10.8 3.6 15.1
1.7 13.0 4.3 18.1
27.25 - 2.0 5.7 1.9 8.0
1.8 6.5 2.2 9.1
36.00 - 0.7 1.7 0.6 2.4
0.6 2.0 0.7 2.8
46.00 - 0.0 0.0 0.0 0.0
0.0 0.1 0.0 0.1

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

The study was performed according to the OECD Guideline 476 and EU-Method B.17 without deviations and considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. Precipitation occurred in experiments conducted at 100 µg/mL. With and without metabolic activation the test material did not induce significant increases in gene mutations in mammalian cells. The test item is considered to be non-mutagenic in this assay.
Executive summary:

The test item, 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol, was assayed for its mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+tk- to tk-tk- (Riach, 2013). The test substance was formulated in acetone. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). The study was designed to be consistent with ICH Guidelines,OECD Guideline No. 476 and EC Directive 2000/32/EC B.17. The study also meets the requirements of the United States and Japan. Preliminary cytotoxicity tests showed that 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol was of a moderate to high order of toxicity, reducing cell growth over the range 1 to 33.3 µg/mL in the absence of S9 mix (4 h exposure period), 3.3 to 100 µg/mL in the presence of S9 mix (4 h exposure period) and over the range 0.3 to 10 µg/mL in the absence of S9 mix (24 h exposure period).

Four independent mutation assays were conducted (assay No. 1 - Absence of S9 – treatment time 4 h – Concentrations (µg/mL): 1.00, 1.75, 3.25, 5.50, 8.50; assay No. 2 - Presence of S9 – treatment time 4 h – Concentrations (µg/mL): 4.75, 8.50, 13.50, 19.75, 27.25; assay No. 3 - Absence of S9 – treatment time 24 h – Concentrations (µg/mL): 1.0, 1.9, 3.1, 4.6, 6.4; assay No. 4 - Presence of S9 – treatment time 4 h – Concentrations (µg/mL): 4.75, 8.50, 13.50, 19.75 - + Concentrations included in the final assessment). In assay 1, the three higher concentrations (12.25 16.75 and 22.00 μg/mL) were all too toxic for assessment.

Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘small’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate. Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, all experiments were tested for dose-related trends in mutant fraction. No reproducible biologically relevant increase in mutant fraction was obtained in either the absence or the presence of S9 mix. (It was noted that small increases considered to be of no biological relevance were consistently obtained in the 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol treatment groups). Results were obtained at a definitive level of toxicity in all 4 mutation assays.

In conclusion, 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix when tested in acetone at concentrations extending into the toxic range.