2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation assay

Dimercapto-1,8-dioxa-3,6-octane (DMDO) was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy (De Marzi, 2016). The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone. DMDO was used as a solution in dimethylsulfoxide (DMSO).

DMDO was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Toxicity was observed with WP2 uvrA tester strain, at the two highest dose levels, in the absence of S9 metabolism and with TA1537, at the highest concentration tested, both in the absence and presence of S9 metabolic activation.

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, TA98, TA100: ±S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA1537: ±S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- WP2 uvrA: -S9: 2500, 1250, 625, 313 and 156 µg/plate /+S9: 5000, 2500, 1250, 625 and 313 µg/plate

No precipitation of DMDO was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Slight toxicity was observed with WP2 uvrA tester strain, at the highest dose level, in the absence of S9 metabolism, and with TA1537, at the highest concentration tested, in the absence and presence of S9 metabolic activation. No relevant increase in the number of revertant colonies was observed, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The dose-range was slightly modified to take into account the toxicity results of Main Assay I. The test item was assayed at the following dose levels:

- TA1535, TA98, TA100: ±S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA1537: ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- WP2 uvrA: -S9: 2500, 1250, 625, 313, 156 and 78.1 µg/plate /+S9: 2500, 1250, 625, 313 and 156 µg/plate.

No precipitation of DMDO was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. An increasing and dose-related toxic effect was observed with almost all tester strains, in the absence and presence of S9 metabolism. Only with WP2 uvrA, a slight toxic effect was observed at the highest or two highest dose levels, in the presence and absence of S9 metabolism, respectively. In order to have a sufficient number of analysable concentrations, an additional experiment (Main Assay III), using the pre-incubation method, was performed with the following tester strains and dose-levels:

- TA1535, TA100: -S9: 625, 313, 156, 78.1 and 39.1 / +S9 1250, 625, 313, 156 and 78.1 µg/plate

- TA98: ±S9: 1250, 625, 313, 156 and 78.1 µg/plate

A confirmatory slight toxicity was noticed at the highest dose level tested, with the three tester strains. No relevant increase in the number of revertant colonies was observed, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

DMDO did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that DMDO does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

DMDO was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method (Bisini, 2017). The test item was found to be soluble in DMSO. A concentration of 1820 µg/mL corresponding to 10 mM, which is the maximum dose level stated in the Study Protocol, was selected as the top dose level to be used. In the preliminary cytotoxicity test, both in the absence and presence of S9 metabolic activation, the test item was assayed at this concentration and at a wide range of lower dose levels: 910, 455, 228, 114, 56.9, 28.4, 14.2 and 7.11 µg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived to treatment at 1820 µg/mL, moderate toxicity was observed at 910 µg/mL, while slight reduction in Relative Survival (RS) was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived to treatment at the two highest concentrations, severe toxicity was noted at the next two lower concentrations (RS= 4 and 5%), moderate toxicity was observed at 114 µg/mL (RS=19%), while slight or no reduction in RS was noted over the remaining dose levels. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived to treatment at the highest dose level, marked toxicity was noted at the next lower concentration of 910 µg/mL, reducing survival to 10% of the negative control, while no relevant toxicity was observed over the remaining concentrations tested. Based on the results obtained in the preliminary trial, three independent assays for mutation at the TK locus were performed using the following dose levels:

Main Assay I (+S9, 3 hour treatment): 1000, 750, 500, 200, 80.0 and 32.0 µg/mL.

Main Assay I (-S9, 3 hour treatment): 900, 692, 533, 410, 315 and 242 µg/mL.

Main Assay II (-S9, 3 hour treatment): 900, 750, 625, 521, 434 and 362 µg/mL.

Main Assay III (-S9, 24 hour treatment): 120, 92.3, 71.0, 54.6, 42.0 and 32.3 µg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. After three hours of treatment in the presence of S9 metabolism or in its absence using the long treatment time, no relevant increase in mutant frequencies was observed at any concentration tested. In the absence of S9 metabolism using the short treatment time, an increase in mutant frequencies, which exceeded the Global Evaluation Factor (GEF), was observed at 750 µg/mL. However, this concentration elicited a RTG value of 13%. Since care should be taken when interpreting mutation results only found at high level of cytotoxicity, a second experiment for this treatment series was performed. A narrowrange of concentrations was used in order to focus on the dose levels most likely to induce mutagenic effects. No increase in mutant frequencies above the GEF was observed at any concentration tested.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that DMDO does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Micronucleus assay

DMDO was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment both in the absence and presence of S9 metabolic activation (Bisini, 2017). Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the absence and presence of S9 metabolism. The harvest time of 31 hours, corresponding to approximately 2.0 cell cycles, was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism, using continuous treatment until harvest at 31 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). For the first main experiment, the highest concentration of 1820 µg/mL was used, corresponding to 10 mM, the upper limit to testing indicated in the Study Protocol. The following lower concentrations were assayed: 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL. Based on the toxicity results obtained, concentrations of 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL were used for the second main experiment. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronuclei in binucleated cells. On the basis of the cytotoxicity of the test item, calculated by the cytokinesis-block proliferation index (CBPI), the following concentrations were selected for the scoring of micronuclei:

Experiment No.:	S9	Treatment time (hours)	Harvest time (hours)	Concentration (µg/mL)
1	-	3	31-32	539, 360 and 240
1	+	3	31-32	1820, 539 and 160
2	-	31	31	47.3, 31.6 and 21.0

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any concentration, in any treatment series. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine indicating the correct functioning of the test system and all results were within the distribution of historical negative control data. It is concluded that DMDO does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 01 July 2016; End of experimental phase: 01 August 2016; Study completion: 31 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

The E.coli used for this study was the strain WP2 uvrA.

Details on mammalian cell type (if applicable):

Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement: No Growth onMinimal plates+Biotin; Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates; Growth onMinimal plates+Tryptophan.
- uvrA, uvrB: Sensitivity to UV irradiation.
- rfa: Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from rats pre-treated with Phenobarbital and 5,6-Benzoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate (based on solubility test).

Main Assay I:
- TA1535, TA98, TA100: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA1537: ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- WP2 uvrA: - S9: 2500, 1250, 625, 313 µg/plate and 156 / +S9: 5000, 2500, 1250, 625 and 313 µg/plate

Main Assay II:
- TA1535, TA98, TA100: ± S9: 5000, 2500, 1250, 625 and 313 µg/plate
- TA1537: ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- WP2 uvrA: -S9: 2500, 1250, 625, 313, 156 and 78.1 / + S9: 2500, 1250, 625, 313 and 156 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Details on test system and experimental conditions:

The preliminary toxicity test and the first experiment were performed using a plate-incorporation method. The second and third experiments were performed using a pre-incubation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al. 1981); Regression line.

Key result

Species / strain:

other: S.typhyimurium TA1535, TA1537, TA98 and TA100; E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item did not induce two-fold increases in the number of revertant colonies, in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

Dimercapto-1,8-dioxa-3,6-octane (DMDO) was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone. DMDO was used as a solution in dimethylsulfoxide (DMSO).

DMDO was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Toxicity was observed with WP2 uvrA tester strain, at the two highest dose levels, in the absence of S9 metabolism and with TA1537, at the highest concentration tested, both in the absence and presence of S9 metabolic activation.

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

- TA1535, TA98, TA100: ±S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA1537: ±S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- WP2 uvrA: -S9: 2500, 1250, 625, 313 and 156 µg/plate /+S9: 5000, 2500, 1250, 625 and 313 µg/plate

No precipitation of DMDO was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Slight toxicity was observed with WP2 uvrA tester strain, at the highest dose level, in the absence of S9 metabolism, and with TA1537, at the highest concentration tested, in the absence and presence of S9 metabolic activation. No relevant increase in the number of revertant colonies was observed, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The dose-range was slightly modified to take into account the toxicity results of Main Assay I. The test item was assayed at the following dose levels:

- TA1535, TA98, TA100: ±S9: 5000, 2500, 1250, 625 and 313 µg/plate

- TA1537: ± S9: 5000, 2500, 1250, 625, 313 and 156 µg/plate

- WP2 uvrA: -S9: 2500, 1250, 625, 313, 156 and 78.1 µg/plate /+S9: 2500, 1250, 625, 313 and 156 µg/plate.

No precipitation of DMDO was observed, at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. An increasing and dose-related toxic effect was observed with almost all tester strains, in the absence and presence of S9 metabolism. Only with WP2 uvrA, a slight toxic effect was observed at the highest or two highest dose levels, in the presence and absence of S9 metabolism, respectively. In order to have a sufficient number of analysable concentrations, an additional experiment (Main Assay III), using the pre-incubation method, was performed with the following tester strains and dose-levels:

- TA1535, TA100: -S9: 625, 313, 156, 78.1 and 39.1 / +S9 1250, 625, 313, 156 and 78.1 µg/plate

- TA98: ±S9: 1250, 625, 313, 156 and 78.1 µg/plate

A confirmatory slight toxicity was noticed at the highest dose level tested, with the three tester strains. No relevant increase in the number of revertant colonies was observed, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

DMDO did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that DMDO does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2016- Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: gene mutation in mammalian cells

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).
This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells.
TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence.
In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK-/- genotype in which little or no TK activity remains.
The cells used, L5178Y TK+/-, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities.
The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers: 3512 and 3488

Test concentrations with justification for top dose:

Main Assay I (+S9, 3 hour treatment): 1000, 750, 500, 200, 80.0 and 32.0 µg/mL.
Main Assay I (-S9, 3 hour treatment): 900, 692, 533, 410, 315 and 242 µg/mL.
Main Assay II (-S9, 3 hour treatment): 900, 750, 625, 521, 434 and 362 µg/mL.
Main Assay III (-S9, 24 hour treatment): 120, 92.3, 71.0, 54.6, 42.0 and 32.3 µg/mL.

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO).
A concentration of 1820 µg/mL corresponding to 10 mM, which is the maximum dose level stated in the Study Protocol, was selected as the top dose level to be used. In the preliminary cytotoxicity test, both in the absence and presence of S9 metabolic activation, the test item was assayed at this concentration and at a wide range of lower dose levels: 910, 455, 228, 114, 56.9, 28.4, 14.2 and 7.11 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 7 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Mutation assay
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system. Preparation of test cell cultures was performed as described in section 4.4. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours), in the absence and presence of S9 metabolism. Equivocal results were obtained in the absence of S9 metabolism, thus this treatment series was repeated inMain Assay II
using a narrow range of concentrations. Since negative results were obtained, an additional experiment (Main Assay III) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete
medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×10^5 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100%nominal relative humidity) to allow for expression of the mutant phenotype.
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10^5 cells/mL.
Plating for 5-trifluorothymidine resistance: After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2×103 cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number ofwells containing large colonies aswell as the number of those containing small colonies were scored.
Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).

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

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In Main Assay I, in the absence of S9 metabolic activation, severe toxicity was noted at the highest dose level, moderate toxicity reducing relative total growth (RTG) to 13% of the concurrent negative control was noted at 750 µg/mL, mild toxicity was observed at 500 µg/mL, while slight or no relevant toxicity was noted at 10the remaining concentrations tested. In the presence of S9 metabolism, treatment with the test item at 900 µg/mL yielded marked toxicity reducing RTG to 7% of the concurrent negative control value, moderate toxicity was noted at the next two lower concentrations, mild toxicity was observed over the remaining concentrations tested.
In Main Assay II, in the absence of S9 metabolic activation using the 3 hour treatment time, the highest dose level selected (900 µg/mL) yielded marked toxicity reducing RTG to 5% of the concurrent negative control value. The next two lower dose levels of 750 and 625 µg/mL yielded moderate toxicity reducing RTG to 19%. Dose-related toxicity was seen over the remaining dose levels tested.
In Main Assay III, in the absence of S9 metabolic activation using the long treatment time, moderate toxicity was observed at the two highest concentrations of 120 and 92.3 µg/mL, reducing RTG to 16 and 24%, respectively. The next lower dose level of 71.0 µg/mL yielded mild toxicity reducing RTG to 35%, while no relevant toxicity was observed over the remaining concentrations tested.

Mutation results:
In Main Assay I, in the presence of S9 metabolism, no relevant increase in mutant frequencies was observed at any concentration tested; in the absence of S9 metabolism a statistically significant increase in mutant frequency, which exceeded the Global Evaluation Factor (GEF), was observed at 750 µg/mL and a linear trend was indicated. However, the observed increase was seen at only one concentration which elicited a RTG value of 13%, thus care should be taken when interpreting this mutation result.
Based on this statement, this treatment series was repeated in Main Assay II. In this second assay, a statistically significant increase in mutant frequency was noted at 625 µg/mL, however this increase was lower than the GEF, thus it was considered of no biological relevance. It must be noted that at the next higher concentration of 750 µg/mL, unacceptable heterogeneity was observed between replicate cultures among mutation plates, thus this concentration has been excluded from the statistical analysis. This variability may be correlated with the different toxic effect
observed in the two replicated cultures on Day 2, confirming that the mutagenic effect is linked to artefacts due to low levels of survival.
In Main Assay III, no increases in mutant frequency were observed at any concentration tested. For the negative and positive controls, the number of wells containing small colonies and those containing large colonieswere reported. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

Conclusions:

It is concluded that DIMERCAPTO-1,8-DIOXA-3,6-OCTANE does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

DIMERCAPTO-1,8-DIOXA-3,6-OCTANE was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The test item was found to be soluble in DMSO. A concentration of 1820 µg/mL corresponding to 10 mM, which is the maximum dose level stated in the Study Protocol, was selected as the top dose level to be used. In the preliminary cytotoxicity test, both in the absence and presence of S9 metabolic activation, the test item was assayed at this concentration and at a wide range of lower dose levels: 910, 455, 228, 114, 56.9, 28.4, 14.2 and 7.11 µg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived to treatment at 1820 µg/mL, moderate toxicity was observed at 910 µg/mL, while slight reduction in Relative Survival (RS) was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived to treatment at the two highest concentrations, severe toxicity was noted at the next two lower concentrations (RS= 4 and 5%), moderate toxicity was observed at 114 µg/mL (RS=19%), while slight or no reduction in RS was noted over the remaining dose levels. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived to treatment at the highest dose level, marked toxicity was noted at the next lower concentration of 910 µg/mL, reducing survival to 10% of the negative control, while no relevant toxicity was observed over the remaining concentrations tested. Based on the results obtained in the preliminary trial, three independent assays for mutation at the TK locus were performed using the following dose levels:

Main Assay I (+S9, 3 hour treatment): 1000, 750, 500, 200, 80.0 and 32.0 µg/mL.

Main Assay I (-S9, 3 hour treatment): 900, 692, 533, 410, 315 and 242 µg/mL.

Main Assay II (-S9, 3 hour treatment): 900, 750, 625, 521, 434 and 362 µg/mL.

Main Assay III (-S9, 24 hour treatment): 120, 92.3, 71.0, 54.6, 42.0 and 32.3 µg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. After three hours of treatment in the presence of S9 metabolism or in its absence using the long treatment time, no relevant increase in mutant frequencies was observed at any concentration tested. In the absence of S9 metabolism using the short treatment time, an increase in mutant frequencies, which exceeded the Global Evaluation Factor (GEF), was observed at 750 µg/mL. However, this concentration elicited a RTG value of 13%. Since care should be taken when interpreting mutation results only found at high level of cytotoxicity, a second experiment for this treatment series was performed. A narrowrange of concentrations was used in order to focus on the dose levels most likely to induce mutagenic effects. No increase in mutant frequencies above the GEF was observed at any concentration tested.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that DIMERCAPTO-1,8-DIOXA-3,6-OCTANE does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study start: 17 May 2016; End of experimental phase: 20 October 2016; Final Report signed: 17 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The test item was assayfor the ability to induce micronuclei in human lymphocytes cultured in vitro, after treatment in the absence and presence of S9
metabolism.

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

Three batches of human whole blood, provided by Biopredic International (France), were
used in this study and had the following characteristics:
Sex: Male
Age: 31 years old
Donor information: healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant: Sodium heparin, 556 IU/mL of whole blood
Collection date: 29-Jun-2016
Batch Number: SAG025A1D009

Sex: Male
Age: 21 years old
Donor information: healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant: Sodium heparin, 556 IU/mL of whole blood
Collection date: 29-Jun-2016
Batch Number: SAG025A1D010

Sex: Female
Age: 22 years old
Donor information: healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant: Sodium heparin, 556 IU/mL of whole blood
Collection date: 07-Sep-2016
Batch Number: SAG025A1D012

The two batches SAG025A1D009 and SAG025A1D010, were pooled and used in Main Experiment 1; the batch number SAG025A1D012 was used in Main Experiment 2.

Cytokinesis block (if used):

the inhibitor of actin polymerisation: cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction, provided by Trinova Biochem GmbH, from Rats pretrerated with Phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

For the first main experiment, the highest concentration of 1820 µg/mL was used, corresponding to 10 mM, the upper limit to testing indicated in the Study Protocol. The following lower concentrations were assayed: 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL.
Based on the toxicity results obtained, concentrations of 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL were used for the second main experiment.

Vehicle / solvent:

Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine 0.080 µg/mL

Details on test system and experimental conditions:

Culture media:
The culture medium for the lymphocytes had the following composition:
RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the
following proportion: 10mL of PHA was added to 500mL of medium.

Preparation of the test cultures and treatment:
Two main experimentswere performed including negative and positive controls. Two cultures were prepared at each test point. Lymphocyte cultures were treated approximately fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.

The composition of the treatment media was as follows:
Treatment medium in the presence of S9 metabolism
Test item or control solution 0.05mL
S9 mix 1.00mL
Culture medium (without PHA) 3.95mL

Treatment medium in the absence of S9 metabolism
Test item or control solution 0.05mL
Culture medium (without PHA) 4.95mL

For the first main experiment, the treatment media was added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were
centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.
For the second main experiment, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Harvesting and slide preparation:
The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this way were dropped onto clean, wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation:
The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI = mononucleated + (2×binucleated) + (3×multinucleated) / total number of cells counted

where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells.
CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed. The highest concentration for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.

The percentage cytotoxicity was evaluated according to the following formula:
% Cytotoxicity = 100 - [100 x (CBPIt-1/CBPIc -1)]

where:
t = test item treated culture
c = vehicle /solvent control culture

The highest concentration for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50 % and 60 %. In the absence of cytotoxicity, the highest treatment level is selected as the highest concentration for scoring.
Two lower concentrations were also selected for the scoring of micronuclei. For the three selected concentrations, for the solvent and for the positive controls (Cyclohosphamide),
1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was
observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

The criteria for identifying micronuclei were as follows:
1. the micronucleus diameter was less than 1/3 of the nucleus diameter;
2. the micronucleus diameter was greater than 1/16 of the nucleus diameter;
3. no overlapping with the nucleus was observed;
4. the aspect was the same as the chromatin.

Evaluation criteria:

Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Criterion for outcome:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the concentrations shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data.

Statistics:

Statistical analysis:
For the statistical analysis, a modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility test
A preliminary solubility trial was performed using DMSO. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity.
The test item was found soluble in DMSO at the concentration of 182 mg/mL. This concentration was chosen since, when added to the treatment medium in the ratio 1:100, it gave a maximum concentration of 1820 µg/mL, corresponding to 10.0 mM which is lower than 2000 µg/mL.
An aliquot of stock solution at the concentration of 182 mg/mL, added to culture medium in the ratio of 1:100, gave a clear formulate, with mild precipitate which disappeared after ten minutes at 37°C. Based on these results, the final concentration of 10 mM in DMSO was selected as the highest concentration to be used in the cytogenetic assay.

Experimental design
Two main experiments were performed. Based on the results obtained in the preliminary solubility test, in Experiment 1, the test item was assayed at the following concentrations: 1820, 1210,809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL. In Experiment 2, using the continuous treatment in the absence of S9 metabolism, the concentration range was slightly modified based on cytotoxicity results obtained in the previous experiment. The following concentrations were assayed: 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL.
Appropriate negative and positive control cultures were included in the experiments. Using the short treatment time (Experiment 1), since tests with and without metabolic activation were done concurrently, positive control cultures were treated only with Cyclophosphamide at the concentrations of 20.0 and 15.0 µg/mL. Using the long treatment time (Experiment 2), in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the concentrations of 80.0, 40.0 and 20.0 ng/mL.

Selection of concentrations for scoring
The CBPI was calculated for each of the treatment series and the results are presented in Table 1.
Using the short treatment time in the absence of S9 metabolism, dose related cytotoxicity was observed reaching the adequate level of 54% at 539 µg/mL.
Following treatment in the presence of S9 metabolism, 59% of cytotoxicity was observed at the highest concentration tested. Mild to slight cytotoxicity was observed at lower concentra- tions down to 360 µg/mL, while no relevant cytotoxicity was observed over the remaining concentrations tested.
In Experiment 2, using the continuous treatment in the absence of S9 metabolism, dose related, moderate to severe, cytotoxicity was observed at higher concentrations reaching 53% at 47.3 µg/mL. No remarkable cytotoxicity was observed over the remaining concentration range.
No precipitation of the test item was observed at the end of treatment, in any treatment series.
On the basis of these results, the concentrations selected for scoring of micronuclei were as follows:

Experiment S9 Treatment time Harvest time Concentration
No.: (hours) (hours) (µg/mL)
1 - 3 31-32 539, 360 and 240
1 + 3 31-32 1820, 539 and 160
2 - 31 31 47.3, 31.6 and 21.0

In agreement with the criteria stated in the Study Protocol, for each treatment series, the max- imum concentration selected for scoring induced adequate level of cytotoxicity (50%-60%), the lowest concentration was chosen in order to have no cytotoxicity, while the intermediate concentration was evenly spaced between the two.
Positive control treatment levels selected for the scoring were as follows:

Experiment S9 Treatment Harvest Concentration
No.: time (hours) time (hours) (µg/mL)
1 + 3 31-32 Cyclophosphamide 20.0
2 - 31 31 Colchicine 80.0

Osmolality and pH results
Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any concentration, in the absence or presence of S9 metabolism. Results are presented in Table 2.

Assay results
The number of binucleated cells with micronuclei is presented in Table 3.
Following treatment with the test item, no remarkable increase in the incidence of micronuc- leated cells over the control value was observed in the absence or presence of S9 metabolism, at any concentration, in any experiment.
Marked increases in the incidence of micronucleated cells were observed following treat- ments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

Analysis of results
The statistical significance of the recorded number of cells bearing micronuclei together with the results of linear trend analysis are shown in Table 4.
Results show that the incidence of micronucleated cells of the negative control was within the distribution range of the historical control values.
Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.
No statistically significant increase in the incidence of micronucleated cells was observed at any concentration, in any treatment series and all the results were within the normal distribution range of historical control data. No concentration related increase of cells bearing micronuclei was observed in any experiment.
On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in human lymphocytes after in vitro treatment.
A summary of the results is presented in Table 5, giving the incidence of micronucleated cells and the cytotoxicity for each test point.

Conclusions:

DIMERCAPTO-1,8-DIOXA-3,6- OCTANE does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

DIMERCAPTO-1,8-DIOXA-3,6-OCTANE was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment both in the absence and presence of S9 metabolic activation. Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the absence and presence of S9 metabolism. The harvest time of 31 hours, corresponding to approximately 2.0 cell cycles, was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism, using continuous treatment until harvest at 31 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). For the first main experiment, the highest concentration of 1820 µg/mL was used, corresponding to 10 mM, the upper limit to testing indicated in the Study Protocol. The following lower concentrations were assayed: 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL. Based on the toxicity results obtained, concentrations of 1210, 809, 539, 360, 240, 160, 107, 71.0, 47.3, 31.6, 21.0 and 14.0 µg/mL were used for the second main experiment. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronuclei in binucleated cells. On the basis of the cytotoxicity of the test item, calculated by the cytokinesis-block proliferation index (CBPI), the following concentrations were selected for the scoring of micronuclei:

Experiment No.:	S9	Treatment time (hours)	Harvest time (hours)	Concentration (µg/mL)
1	-	3	31-32	539, 360 and 240
1	+	3	31-32	1820, 539 and 160
2	-	31	31	47.3, 31.6 and 21.0

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any concentration, in any treatment series. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine indicating the correct functioning of the test system and all results were within the distribution of historical negative control data. It is concluded that DIMERCAPTO-1,8-DIOXA-3,6-OCTANE does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available