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EC number: 210-871-0 | CAS number: 624-92-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A lot of in vitro studies were performed on DMDS. No positive mutagenic response was observed in the Ames test. However, DMDS did not induce structural chromosome aberrations in cultured human lymphocytes at non toxic concentrations but it can not be excluded that DMDS can react as a clastogen at very toxic concentrations, both in the absence and in the presence of metabolic activation (OECD 473). The in vitro mammalian cell gene mutation assay showed inconclusive results.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine locus (Salmonella) and tryptophane locus (Escherichia)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254
- Test concentrations with justification for top dose:
- The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate in the initial toxicity-mutation assay and 50, 150, 500, 1500 and 5000 µg per plate in the confirmatory mutagenicity assay.
- Vehicle / solvent:
- Dimethyl sulphoxide
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: With S9: 2-aminoanthracene, 1 µg/plate for all Salmonella strains, 10µg/plate for E. coli. Without S9: TA 98, 2-nitrofluorene (1 µg/plate); TA100 and TA 1535, sodium azide (1 µg/plate); TA 1537, 9-aminoacridine (75 µg/plate); E. coli, MMS (1000 µg/plate)
- Details on test system and experimental conditions:
- DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn);
EXPERIMENTS
- Initial toxicity-mutation assay: in all strains, with or without S9 mix ; 8 dose-levels (2 plates/dose level)
- Confirmatory mutagenicity assay: in all strains, with or without S9 mix ; 5 dose-levels (3 plates/dose level)
METHOD OF APPLICATION: Direct plate incorporation method: for preliminary both experiments
DURATION
- Exposure duration: 48-72H - Evaluation criteria:
- Reproducible increase in the number of revertant colonies (2-fold for TA98/TA100 and WP2 uvrA, 3-fold for TA 1535/TA 1537) compared with vehicle controls in any strain at any dose-level and/or evidence of a dose-relationship.
Reference to historical data and consideration to biological relevance may also be taken into account. - Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to limit concentrations recommended by the test guideline
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING STUDY:
- Results on solubility: dimethyl disulphide formed a soluble and clear solution in dimethyl sulfoxide (DMSO) at approximately 500 mg/mL, the highest concentration tested.
- Results on cytotoxicity: In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor appreciable toxicity was observed. - Conclusions:
- The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Dimethyl disulphide did not cause a positive response in either the presence or absence of Aroclor-induced rat liver S9.
- Executive summary:
Dimethyl disulfide, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed using the plate incorporation method. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate in the initial toxicity-mutation assay and 50, 150, 500, 1500 and 5000 µg per plate in the confirmatory mutagenicity assay. No positive mutagenic response was observed. Neither precipitate nor appreciable toxicity was observed. Dimethyl disulfide was concluded to be negative in the Bacterial Reverse Mutation Assay
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1983
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- n/a
- Species / strain / cell type:
- primary culture, other: Human Lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 derived from adult male Wistar rats (Aroclor 1254 induced rat liver).
- Test concentrations with justification for top dose:
- 0; 3.7; 11.1; 33.3; 100; 300 µg/ml
- Vehicle / solvent:
- The test article (dissolved in Dimethyl sulfoxide (DMSO)) was soluble in culture medium at a maximum concentration of 1 mg/mL
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Without S9: mitomycin C (MMC) 0.05 µg/mL. With S9: cyclophosphamide (CP) 25 µg/mL
- Details on test system and experimental conditions:
- - Preliminary Cytotoxicity Assay: The dose levels used in the chromosome aberration assay were established on the basis of the results of a preliminary toxicity test carried out with 6 concentrations of the test substance (ranging from 0.5 to 1000.0 µg/ml), both in the absence and in the presence of the metabolic activation system (S-9 mix). The highest concentration for the toxicity test was determined by the limit of the solubility of the test substance in the tissue culture medium (RPMI 1640 medium supplemented with heat-inactivated foetal calf serum, 100 units penicillin/mL, 100 µg streptomycin/mL, 2 mM L-glutamine and 25 µl phytohaemagglutinin/ml)
- Cytogenetic Assay:
* Cell Treatment
After 48 h of incubation, the cultures were centrifuged at 800 rpm (100 g) and the supernatant removed. The cell pellets were resuspended in 4.5 ml tissue culture medium supplemented with 20 mM HEPES (and 10% S-9 mix, for the test with metabolic activation) and containing 50 µl of the appropriate test solutions. The final concentrations of the test substance in the culture medium were: 0.5, 1.4, 4.1, 12.3, 37.0, 111.1, 333.3 and 1000.0 µg/ml.An untreated culture and a culture receiving 50 µl of DMSO served as negative controls. For each concentration of the test substance and for the controls one culture was used. Without S9, the cultures were incubated in closed tubes for another 24 hours including a 2 hour colcemid treatment at 37°C in humidified air containing 5% CO2. With S-9 mix, the exposure of the cells to the test substance was reduced to only 2 hours, because of the toxicity of the S-9 mix for the cells. After the 2 hour incubation period, the cultures were centrifuged, the supernatant removed, the cells washed with phosphate- buffered saline (pH 7.4) and subsequently supplied with 4.5 ml freshly prepared culture medium. The cells were incubated for a further 22 hours (including a 2 hour colcemid treatment.
* Cell harvesting: Two hours before the end of the total incubation period the cells were arrested in the metaphase stage of the mitosis by the addition of colcemid (final concentration: 0.1 µg/ml medium). The cells were harvested by low speed centrifugation, treated for 15 minutes at 37°C with a hypotonic solution (0.075 M KCl), fixed three hours with a 3:1 mixture of methanol and glacial acetic acid, and transferred to clean microscope slides. Two slides were prepared from each culture. The slides were stained for 10 minutes in a 2% solution of Giemsa, rinsed in water, dried and mounted in DePeX. In each culture 1000 stimulated lymphocytes were examined (500 from each slide) to determine the mitotic index (percentage of cells in mitosis
* Metaphase analysis: From each culture, 100 well-spread metaphases (each containing 46 chromosomes) were analysed by microscopic examination for a wide range of structural chromosome aberrations (gaps, breaks, fragments, dicentrics, exchanges etc.) and other anomalies (endoreduplication, polyploidy), according to the criteria recommended by Savage (1975). - Evaluation criteria:
- The major criterion to designate the results of a chromosome aberration test as positive is a dose related, statistically significant increase in the number of cells with structural chromosome aberrations. However, a clear dose response relationship can be absent because the yield of chromosome aberrations can vary markedly with post treatment sampling time of an asynchronous population and because increasing doses of clastogens can induce increasing degrees of mitotic delay. A test substance producing neither a dose related, statistically significant increase in the number of cells with structural chromosome aberrations, nor a statistically significant and reproducible positive response at any of the doses is considered non-clastogenic in this system.
- Statistics:
- Fischer's exact probability test.
- Species / strain:
- primary culture, other: Human Lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- clearly toxic at >= 300 µg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Dimethyl disulphide did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at non toxic concentrations, both in the absence and in the presence of the S-9 mix. At the very toxic concentration of 300.0 µg/ml, both in the absence and in the presence of the S-9 mix, the test substance induced a statistically significant increase in the number of cells with structural chromosome aberrations.
The positive control substances, mitomycin C and cyclophosphamide, induced the expected increase in the incidence of structural chromosome aberrations. - Conclusions:
- Dimethyl disulphide (DMDS) did not induce structural chromosome aberrations in cultured human lymphocytes at non toxic concentrations but it can mot be excluded that DMDS can react as a clastogen at very toxic concentrations, both in the absence and in the presence of the S-9 mix, under the conditions used in the present assay.
- Executive summary:
The potential of dimethyl disulphide (DMDS) to induce structural chromosome aberrations in human lymphocytes was evaluated according to OECD guideline in compliance with the Principles of Good Laboratory Practice. DMDS was tested in one experiment, with and without a metabolic activation system. The lymphocytes cultures were exposed to positive and negative controls or DMDS at concentrations of 3.7, 11.1, 33.3, 100 and 300 µg/ml. The cultured cells were exposed for 2 hours with S9 mix and continuously until harvest without S9 mix. All cells were harvested 24H after initiation of the treatment. DMDS did not induce structural chromosome aberrations in cultured human lymphocytes at non toxic concentrations but it can not be excluded that DMDS can react as a clastogen at very toxic concentrations, both in the absence and in the presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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:
- 1984
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HGPRT locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 derived from adult male Wistar rats (Aroclor 1254 induced rat liver)
- Test concentrations with justification for top dose:
- First assay : 0.46, 1.37, 4.12, 12.3, 37.0, 111, 333 and 1,000 mg/l
Second assay: 4.12, 12.3, 37.0, 74.1, 111, 333, 667 and 1,000 mg/l - Vehicle / solvent:
- DMDS (dissolved in DMSO) was soluble in culture medium at a maximum concentration of 1 mg/mL
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Without S9: Ethylmethanesulfonate 0.2 ml/L . With S9: Dimethylnitrosamine 2 or 4 ml/L.
- Details on test system and experimental conditions:
- The dose levels used in the HGPRT assay were established on the basis of the results of a preliminary solubility test. A final concentration of 1,000 µg/ml was chosen as highest concentration for the HGPRT assays.
For the HGPRT-assay, aliquots of 1.8 x 10e6 CHO tells were added to a number of 75 cm2 tissue culture flasks containing 10 ml Ham's F-12 growth medium. The cells were incubated for approximately 20 hours in a humidified incubator at 37°C in air containing 5% CO2. On the day following seeding, the cells were exposed to the test substance, both in the absence and in the presence of a metabolic activation system (S-9 mix).
In the absence of the S-9 mix, the cells were exposed to the test substance for 3 hours according to the following procedure. On the day of exposure, the tissue culture medium was removed and replaced by 9.9 ml Ham's F-12 containing gentamicin (50 mg/l) and L-glutamine (2 mM). One hundred µl of each of the test solutions were added to the culture medium. The final concentrations of the test substance in the culture medium selected for the exposure of the cells were: a) first assay : 0.46, 1.37, 4.12, 12.3, 37.0, 111, 333 and 1,000 mg/l, b) second assay: 4.12, 12.3, 37.0, 74.1, 111, 333, 667 and 1,000 mg/l.
In the presence of the S-9 mix, the cells were exposed to the test substance according to the following procedure. On the day of exposure, the tissue culture medium (Ham's F-12 medium supplemented with 10% heat-inactivated foetal calf serum, 50 µg gentamicin/mL and 2 mM L-glutamine) was removed and replaced by Ham's F-12 culture medium containing gentamicin (50 mg/1), L-glutamine (2 mM) and 10% (v/v) S-9 mix. The final concentrations of the test substance in the culture medium selected for the exposure of the cells were:
a) first assay : 0.46, 1.37, 4.12, 12.3, 37.0, 111, 333 and 1,000 mg/l,
b) second assay: 4.12, 12.3, 37.0, 74.1, 111, 333, 667 and 1,000 mg/l.
For each concentration of the test substance and for the controls, one culture was used. After the 3-hour incubation period at 37°C the medium was removed, the cells were washed with phosphate-buffered saline (pH 7.4) and supplied with 10 ml growth medium. Subsequently, the cultures were incubated for an additional 18-21 hours in a humidified incubator at 37°C in air containing 5% C02.
Cytotoxicity of the test substance was determined by measuring the colony-forming ability (cloning efficiency) of the CHO cells after the treatment period in the two independent HGPRT assays.
The two independent HGPRT-assays were carried out with single cultures for each concentration of the test substance and for the negative and positive controls. - Evaluation criteria:
- The following criteria were used to evaluate the data obtained in the HGPRT assay (Li et al. 1987)
a) the survival (absolute cloning efficiency) of the negative controls should not be less than 50%,
b) the mean mutant frequency of the negative controls should fall within the range of 0-20 6-TG resistant mutants per 10e6 clonable cells,
c) the positive controls must induce a response of a magnitude appropriate for the mutagen under the experimental conditions applied,
d) the highest test substance concentration should, if possible, result in a clear cytotoxic response (e.g. 10-30% of the relative initial survival).
Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% toxicity is considered to be an artifact and not indicative of genotoxicity. Genotoxicity of the test substance was evaluated using the following criteria (Li et al. 1987):
a) a concentration-related increase in mutant frequency,
b) a reproducible positive response for at least one of the test substance concentrations (e.g. the mean mutant frequency should be more than 20 mutants per 10e6 clonable cells). - Statistics:
- Exact statistical analysis is difficult because the distribution of the number of mutant colonies depends on assumptions of homogeneous variance, normal distribution, and the complex processes of cell growth and cell death after treatment with a test substance or a positive control (Li et al. 1987). Therefore, evaluation of the data obtained with the HGPRT assay was made on a case by case basis using the above described criteria, rather than by statistical analysis.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 74.0-1000 µg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The actual concentrations of DMDS in culture medium were much lower than the target concentrations (see attached graph). Recovery experiments showed that about 50% of DMDS was lost directly on incubation (presumably by evaporation). Furthermore, during incubation an additional amount of 25% of DMDS is lost (presumably reactions with constituents of the incubation).
In the absence of a metabolic activation system, the highest concentration (1,000 mg/l) showed an increased mutant frequency, only in the first HGPRT assay. At that concentration the test substance was highly toxic to the cells: the absolute initial cloning efficiency was reduced to about 22%. Furthermore, at this concentration droplets attached to the bottom of the tissue culture flask were observed. At lower concentrations (667, 333, 111 and 74.0 mg/l) which were still highly toxic, and at non-toxic concentrations (37.0 mg/l and lower), the mutant frequency did not differ clearly from that of the negative controls, in both independent assays. In view of these observations, the non-reproducible increase of the mutant frequency, at the highest concentration used, is not considered to be of biological significance.
In the presence of a metabolic activation system, slight increases in mutant frequency were observed at several concentrations, both in the first (0.46, 12.3, 37.0, and 1,000 mg/l) and in the second assay (1,000 and 667 mg/l). Such increases occurred both at clearly toxic concentrations (absolute initial cloning efficiency of about 20%) and at non-toxic concentrations of the test substance. The increases in mutant frequency were not concentration-related.
The positive control substances, EMS (in the absence of the S-9 mix) and DMN (in the presence of the S-9 mix), showed the expected increases in mutant frequency. - Conclusions:
- No conclusive evidence for a genotoxic effect of DMDS was found in cultured CHO cells, under the conditions used in the HGPRT assay.
- Executive summary:
Dimethyldisulfide (DMDS) was examined for its potential to induce point mutations in the HGPRT-locus of cultured Chinese hamster ovary (CHO) cells, both in the absence and in the presence of a metabolic activation system (S-9). The test was conducted in compliance with OECD guideline 476. The dose levels used in the HGPRT assay were established on the basic of the results of a preliminary solubility test. Both in the absence and in the presence of a metabolic activation system (S-9 mix), the cells were exposed for 3 hours to 8 concentrations of DMDS: 0.46, 1.37, 4.12, 12.3, 37.0, 111, 333 and 1,000 mg/l in the 1st assay and 4.12, 12.3, 37.0, 74.0, 111, 333, 667 and 1,000.0 mg/l in the 2nd assay. Ethylmethanesulfonate (in the absence of the S-9 mix) and dimethylnitrosamine (in the presence of the S-9 mix) were used as positive controls, while the vehicle (DMSO) and culture medium served as negative controls.
In the absence of the S-9 mix, DMDS induced neither a concentration-related increase in the mutant frequency nor a reproducible positive response at one of the test concentrations. In the presence of a metabolic activation system, DMDS induced a slight increase in mutant frequency at several concentrations, in both HGPRT assays. These increases were neither concentration-related nor clearly reproducible. In both HGPRT assays, the test substance appeared to be highly toxic to CHO cells at a concentration range from 74.0-1,000 mg/l. The actual concentrations of DMDS in culture medium were much lower than the target concentrations. The positive control substances induced the expected increase in the mutant frequency.
No conclusive evidence for a genotoxic effect of DMDS vas found in cultured CHO tells, under the conditions used in the HGPRT assay
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Version / remarks:
- 1986
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
- Species / strain / cell type:
- primary culture, other: Rat hepatocytes
- Metabolic activation:
- not applicable
- Test concentrations with justification for top dose:
- * Cytotoxicity studies:
- 1st study : 1 - 5 - 10 - 100 and 200 µg/ml
- 2nd study : 5 - 10 - 50 - 100 - 150 - 200 - 250 and 300 µg/ml
* Genotoxicity studies:
- 1st study: 1- 5 - 10- 50 - 100 and 200 µg/ml
- 2nd study : 1 - 10 - 50 - 100 - 200 and 300 µg/ml - Vehicle / solvent:
- Dimethyl sulfoxide (DMSO). DMDS was soluble in culture medium at a maximum concentration of 100 µg/mL.
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- yes
- Remarks:
- Pyrene 1 µM
- Positive controls:
- yes
- Positive control substance:
- other: 7,12-DMBA (10 µM) and 2-aminofluorene (0.1 and 0.5 µM)
- Details on test system and experimental conditions:
- - Cytotoxicity evaluation:
The test compound cytotoxicity was assessed for both DNA repair studies at the end of the treatment:
. by optical microscopic observation of the cell cultures,
. by measurement of the lactate dehydrogenase (LDH) activity.
- Incubation:
Each concentration of Dimethyldisulfide was tested in triplicate. After 18 to 20 hours in a 95% air and 5% C02 humidified 37°C incubator, hepatocytes were washed with 2 ml of WME and observed under a microscope. Each coverslip was then washed with WME and immersed in 2 ml of 1% hypotonic sodium citrate, inducing a swelling of nuclei and a better quantification of nuclear grains. Finally, the cells were fixed in three 30-minute changes of ethanol and acetic acid (3:1), air-dried and mounted cell surface up on glass slides.
- Autoradiography:
Autoradiographs were prepared by dipping slides in a photographic emulsion then developed. Slides were stained in hematoxylin-phloxin.
- Slide assessment:
Grain courts were performed using an Artek electronic counter, connected to a microscope. For each cell, following nuclear grain court, cytoplasmic count was performed on 3 areas of the same size as the nucleus and adjacent to it. - Evaluation criteria:
- The test compound is considered positive when the mean nuclear grain court is statisticaly greater than that of the control, the mean net nuclear grain court is above 3 grains per nucleus, and the percentage of treated cells in repair is significantly different from that of the controls. In addition, the effect must be shown to be reproducible between experiments.
- Statistics:
- Not appropriate
- Species / strain:
- primary culture, other: Rat hepatocytes
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- >= 100 µg/ml. IC50 evaluated by LDH release: 98 µg/ml (2nd study)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- In both studies, the mean incorporation of tritiated thymidine in the nuclei of cells treated with DMDS at concentrations of 50, 100 and 200 µg/ml, was similar to that observed in control cells (solvent or untreated), and was lower than that observed in cytoplasms.
The positive controls (DMBA and 2-AF), run in parallel induced incorporation of many nuclear grains at both concentrations, thus indicating an intense DNA repair synthesis due to a very active metabolism of hepatocytes, while pyrene control induced rates of incorporation of tritiated thymidine similar to chose obtained in control cells (solvent or untreated). - Conclusions:
- DMDS did not induce DNA repair synthesis in the in vitro DNA repair assay at concentrations of 10, 50, 100 and 200 µg/ml throughout 2 independent studies
- Executive summary:
In a study performed according to the OECD Guideline #482, the potential genotoxicity of dimethyl disulphide (DMDS) was assessed in vitro on freshly isolated rat hepatocytes by comparing the incorporation of tritiated thymidine in treated cells with that induced in cells exposed to the solvent (dimethylsulfoxide or DMSO) or to genotoxic agents 2-aminofluorene and 7,12-dimethylbenz(a)anthracene).
DMDS was dissolved in DMSO and diluted at concentrations ranging front 1 to 30 mg/ml. A precipitation of the compound in the culture medium at concentrations from 200 µg/ml upwards was observed.
DMDS was found to be cytotoxic at concentrations of 200, 250 and 300 µg/ml. Cytotoxicity was evaluated by measuring the release of lactate deshydrogenase, which gave an IC50 value of 98 µg/ml. Both DNArepair studies performed at concentrations of 10, 50, 100 and 200 µg/ml did not reveal any induction of unscheduled DNAsythesis in rat hepatocyte primary cultures exposed DMDS. In conclusion, DMDS was not genotoxic to rat hepatocytes in primary culture.
Referenceopen allclose all
Number of revertants per plate (first experiment) (mean of 2 plates) |
||||||||||
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
|||||
Conc. [µg/plate] |
- MA |
+MA |
- MA |
+ MA |
- MA |
+ MA |
- MA |
+MA |
-MA |
+MA |
DMSO |
17 |
19 |
126 |
127 |
16 |
16 |
9 |
5 |
11 |
12 |
1.5 |
21 |
21 |
122 |
131 |
14 |
13 |
6 |
10 |
7 |
13 |
5.0 |
15 |
21 |
101 |
137 |
14 |
18 |
7 |
8 |
8 |
13 |
15 |
19 |
21 |
103 |
142 |
14 |
21 |
2 |
7 |
10 |
9 |
50 |
14 |
24 |
103 |
124 |
10 |
14 |
7 |
6 |
10 |
14 |
150 |
13 |
20 |
95 |
120 |
14 |
17 |
4 |
8 |
11 |
9 |
500 |
20 |
32 |
139 |
137 |
20 |
18 |
9 |
2 |
13 |
12 |
1500 |
17 |
19 |
136 |
131 |
17 |
7 |
4 |
7 |
10 |
10 |
5000 |
14 |
19 |
103 |
138 |
16 |
25 |
7 |
8 |
12 |
13 |
Positive control |
170 |
553 |
426 |
604 |
424 |
70 |
389 |
68 |
52 |
87 |
Table 2: Number of revertants per plate (second experiment) (mean of 3 plates) |
||||||||||
|
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
|||||
Conc. [µg/plate] |
- MA |
+MA |
- MA |
+ MA |
- MA |
+ MA |
- MA |
+ MA |
- MA |
+ MA |
DMSO |
24 |
30 |
119 |
134 |
28 |
15 |
8 |
11 |
14 |
16 |
50 |
18 |
29 |
110 |
137 |
24 |
11 |
6 |
6 |
11 |
13 |
150 |
25 |
29 |
118 |
131 |
32 |
16 |
6 |
5 |
14 |
14 |
500 |
22 |
29 |
113 |
122 |
23 |
14 |
6 |
6 |
14 |
18 |
1500 |
19 |
34 |
107 |
126 |
26 |
15 |
7 |
6 |
15 |
20 |
5000 |
21 |
29 |
113 |
121 |
30 |
18 |
6 |
7 |
16 |
18 |
Positive control |
147 |
458 |
628 |
572 |
478 |
78 |
497 |
53 |
96 |
181 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In a combined in vivo micronucleus assay and in vivo alkaline comet assay (Randazzo 2017), rats exposed by inhalation to DMDS during 3 consecutive days showed no DNA damage and no clastogenic activity.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- September 2016 to August 2017. The study was performed at the request of an EU national competent authority in the frame of Regulation EC 1107/2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Micronucleus assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 7-8 weeks old
- Weight at study initiation:
221 g to 263 g for males and from 155 g to 188 g for females in the Range-Finding Phase
211 g to 272 g for males and from 166 g to 208 g for females in the Definitive Phase
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 2 to 3 per cage by sex in clean, solid bottom cages
- Diet (ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water (ad libitum): reverse osmosis treated (on site) drinking water
- Acclimation period: at least 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5–22.8
- Humidity (%): 35.7-51.5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Vehicle:
- Air
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 500-L or 1000-L glass and stainless steel whole-body exposure chambers
- Method of holding animals in test chamber: individually in standard exposure batteries
- Source and rate of air: in-house compressed air
- Method of conditioning air: HEPA charcoal-filtered, temperature- and humidity-controlled supply air source
- System of generating vapors: Vapors of the test substance were
generated using a bubbler-type vaporization system. A 300-mL or 500-mL gas washing bottle, (Ace Glass, Inc.; Vineland, NJ) was filled with an appropriate amount of liquid test substance. Compressed nitrogen was metered into the inlet stem of the gas washing bottle and bubbled through a fritted disc. Nitrogen was metered to the gas washing bottle using a Coilhose regulator (Model No. 8802K) and controlled using a needle valve and Gilmont rotameter-type flowmeter. Concentrated vapors of the test substance were delivered to the exposure chamber inlet and diluted to the desired atmosphere concentration by mixing with the chamber supply air prior to entering the chamber.
- Temperature, humidity, pressure in air chamber: The mean temperature and mean relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively.
- Air flow rate:
Range-Finder Phase: 214-221 L/min
Definitive Phase: 111-120 L/min
- Air change rate: at least 12 air changes per hour.
- Method of particle size determination: Microdust Pro 880nm aerosol monitoring system. Aerosol content was 0.0 mg/m3 for all high dose groups.
- Treatment of exhaust air: activated-carbon drum prior to passing through the facility exhaust system which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units
TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography/FIG
- Samples taken from breathing zone: yes - Duration of treatment / exposure:
- 6 hours per day
- Frequency of treatment:
- 3 consecutive days
- Post exposure period:
- 2 and 4 hours (DMDS exposed groups)
18–24 hours (positive control group) - Dose / conc.:
- 175 other: ppm (target)
- Remarks:
- 173 ppm (analytical)
- Dose / conc.:
- 350 other: ppm (target)
- Remarks:
- 351 ppm (analytical)
- Dose / conc.:
- 700 other: ppm (target)
- Remarks:
- 679 ppm (analytical)
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, sham-exposed
- Positive control(s):
- Cyclophosphamide monohydrate
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: oral, on study Days 0 and 1 (first and second days of exposure)
- Doses / concentrations: 20 mg/kg/day - Tissues and cell types examined:
- Polychromatic erythrocytes [PCEs] of bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
In order to assess the potential for exposure-limiting toxicity, the high target exposure concentration for the range-finding phase (750 ppm) was selected based on the following previous inhalation studies.
• Four-hour acute inhalation toxicity study in rats: LC50 was 1310 ppm. Mortality was 0/10, 4/10, 4/10, and 9/10 animals in the 847, 1188, 1308, and 1650 ppm groups respectively;
• Four-hour acute inhalation mammalian erythrocyte micronucleus assay in rats: no mortality up to 825 ppm. No significant reductions in the ratio of PCEs to total erythrocytes;
• Four-hour acute inhalation exposure in rats: increased serum glutamic-pyruvic-transaminase immediately after exposure to 600 ppm and increased gamma glutamyltransferase immediately after 500 ppm;
• Six-hour acute neurotoxicity study in rats: mortality was 0/24, 0/24 and 1/24 animals for the 100, 200, and 750 ppm group, respectively;
• Six-hour acute inhalation toxicity study in rats: no mortalities were observed up to 600 ppm. Acute inflammation and degeneration of the transitional and olfactory epithelia and acute inflammation of the respiratory epithelium were noted at 50, 150, 300, and 600 ppm, and degeneration of the respiratory epithelium was noted at concentration of 150 ppm and higher;
• Twenty-four-hour acute inhalation toxicity study: exposure-related degeneration of the olfactory epithelium at exposure levels of 9, 12.5, and 18 ppm and a slight increase in inflammation of the respiratory and olfactory epithelia at 18 ppm;
• Five-day subacute (6 hours/day) inhalation toxicity in rats:10 no mortalities were observed up to 600 ppm. Mean absolute lung and lung/body weights were higher in the 300 and 600 ppm group females. Hyperplasia of the squamous nasal epithelium was noted in = 300 ppm group males and all test substance-exposed group females (= 50 ppm). Hyperplasia of the transitional and respiratory epithelia and degeneration and regeneration of the olfactory epithelium were noted at all test substance exposure concentrations (= 50 ppm) in both sexes.
Range-finding study
In the range finding study, vaporized test substance (DMDS) was administered at 650 anf 750 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2 groups (Groups 2–3) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights and cage food weights were recorded within 4 days of receipt and on the day of randomization (body weights only), on the day the animals were placed into treatment groups (food weights only), and daily throughout the exposure period. The animal found dead had a gross necropsy performed and the carcass was discarded without tissue collection. Clinical pathology parameters (hematology and serum chemistry) were analyzed for all surviving animals within 3 hours following exposure on Study Day 2. Gross necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were collected possible future histopathology. Bone marrow was collected for evaluation of cytotoxicity from all surviving animals at the scheduled euthanasia (Study Day 2; between 2 and 4 hours following the last exposure).
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1–4 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Groups 5–6. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt (body weights only), on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Bone marrow was collected from 5 animals/sex/group from Groups 1–5 at the scheduled necropsy (between 2–4 hours following the last exposure or 18–24 hours following the last dose administration).
Bone marrow was collected from the first 5 of 6 animals in each sex in Groups 1–5 at the time of euthanasia from the right femur of animals anesthetized by isoflurane inhalation and euthanized by exsanguination. Five animals/sex/group from the filtered air control group (Group 1) and test substance-exposed (Groups 2–4) groups were euthanized approximately 2–4 hours following the Study Day 2 exposure, and 5 animals/sex/group from the positive control group (Group 5) were euthanized approximately 18–24 hours following the Study Day 2 dose administration.
DETAILS OF SLIDE PREPARATION:
Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution.
METHOD OF ANALYSIS:
- Bone Marrow Cytotoxicity Analysis
A total of 500 erythrocytes (TE = PCE + NCE) per animal were counted, and the PCE:TE ratios were determined.
- Micronuclei Analysis
For each slide, 500 TE/animal were counted to determine PCE:TE ratios, and 4000 PCEs/animal were scored to determine %MN-PCEs. - Evaluation criteria:
- Data will not be used either in the individual animal data or for statistical evaluation if obtained from study rat with either fewer than 500 PCEs when scoring the %MN-PCEs in immature erythrocytes or a PCE/TE ratio of less than 20% of the filtered air control value.
The filtered air control group mean must lie within the historical control range. The positive control response must be higher than the filtered air control group and be consistent with historical positive control data.
Criteria for Negative Response
Cases that do not clearly fit into the positive or negative criteria may be judged equivocal. In these cases, the Individual Scientist, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results. As a general rule, the biological relevance of any result will be considered first. - Statistics:
- For slides prepared in Range-Finding Phase, the ratio of PCEs to total erythrocytes for the test substance-exposed groups was compared to the filtered air control group using an ANOVA.12 If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett’s test13 was used to compare each test substance-exposed group to the filtered air control group. Statistical significance was assessed at a 95% confidence level (p=0.05).
For the slides prepared in Definitive Phase, the %MN-PCEs and PCE/TE for the filtered air control and test substance-exposed groups were compared using an ANOVA.12 If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett’s test13 was used to compare each test substance-exposed group to the filtered air control group. The Cochran Armitage test, was used for the detection of dose response trends in the test substance-exposed group only. In addition, a comparison of the positive (Group 5) and filtered air control groups was made using a separate ANOVA. Statistical significance was assessed at a 95% confidence level (p=0.05). - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- animals exposed up to the maximal tolerated concentration but no bone marrow cytotoxicity
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
One female was found dead at 750 ppm immediately following exposure on Study Day 0. There were no clinical observations noted for the female found dead. There were no other test substance related effects on survival. A single female in the 750 ppm group was observed with labored respiration on Study Day 2 prior to exposure. Yellow material on the urogenital area and clear material on the ventral neck were observed in the 650 ppm group males and females. In addition, a single 650 ppm group female was observed with vocalization during handling on Study Day 0. There were no other test substance related clinical observations. Test substance-related effects on body weight were noted in all test substance-exposed groups. Significantly lower mean body weights were noted on Study Day 2 in the 650 and 750 ppm group males and females, with only the 650 ppm group females not being statistically significant. Statistically significant lower body weights were also noted in the 750 ppm group males on Study Day 1. Statistically significant mean body weight losses or lower mean body weight gains were noted in the 650 and 750 ppm group males and females throughout the entire exposure period. Test substance-related lower mean food consumption was noted in the 650 and 750 ppm group males and females throughout the exposure period. Test substance-related effects on hematology was noted in all exposed groups. Higher mean RBC, HGB, HCT, and HDW and lower mean absolute eosinophil values (statistically significant in all male groups) were noted in all test substance-exposed group males and females. Statistically significant lower mean neutrophil (percent and absolute) values and higher mean percent lymphocyte values were noted in the 650 and 750 ppm group males. In addition, significantly higher mean platelet values were noted in the 650 ppm group males and 750 ppm group males and females (statistically significant in both male groups). Statistically significant lower mean percent reticulocyte values were noted in the 750 ppm group males and nonstatistically significant lower mean absolute reticulocyte values were noted in the 750 ppm group males. None of the effects on hematology were considered to be evidence of dose-limiting toxicity that would have impacted the dose selection for Definitive Phase. Test substance-related effects on serum chemistry were noted in all exposed group males and females. Test substance-related higher mean albumin, total protein, and globulin values and lower ALT values were noted in all test-substance exposed group males and females. Higher mean creatinine (statistically significant) and lower mean triglyceride values were noted in the 750 ppm group males. In addition, statistically significant higher mean cholesterol values were noted in the 750 ppm group males and females. Test substance-related lower mean phosphorus values were noted in all test substance-exposed group males and was attributed to possible dehydration. Statistically significant lower mean SDH was noted in the 650 ppm group males but was not attributed to the test substance as the change was not present in a dose-responsive manner. None of the effects on serum chemistry were considered to be evidence of dose limiting toxicity that would have impacted the dose selection for Definitive Phase. The test substance did not produce a statistically significant decrease in the PCE:TE ratios at 650 and 750 ppm compared to the filtered air control for male and female rats. Test substance-related lower mean absolute liver weights were noted in the 650 ppm group males and 750 ppm group males and females, higher mean kidney weight relative to body weight was noted in the 750 ppm group females and higher mean relative lung weights were noted in 650 and 750 ppm group males and females. The differences were attributed to the lower final body weights in the test substance-exposed groups.
Based on the achieved exposure concentrations, the concentration of 700 ppm was selected as the high-dose in Definitive Phase, as exceeding this concentration would likely result in dose-limiting toxicity (mortality and/or moribundity).
RESULTS OF DEFINITIVE STUDY
Dimethyl disulfide did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) of bone marrow compared to the vehicle control for male and female rats. No bone marrow cytotoxicity was noted in any male and female rats at any dimethyl disulfide dose level. All animals survived to the scheduled euthanasia. There were no test substance related clinical observations. Statistically significant test substance-related body weight losses or lower mean body weight gains were noted in all test substance exposed group males and in the 700 ppm group females. Test substance-related lower mean food consumption values were noted in all test substance treated group males and females. The group mean values for both %MN PCEs and PCE:TE ratios for the vehicle and positive controls were comparable to the respective historical control ranges demonstrating the sensitivity of the assay. - Conclusions:
- Dimethyl disulfide met the criteria for a negative response for clastogenic activity and/or disruption of the mitotic apparatus under the conditions of this assay. In the absence of medullar toxicity, indirect evidence of the bone marrow exposure was provided by the systemic toxicity, including mortality and decrease of the body weight gain, observed in the range-finding and/or definitive studies and consistent with the available acute toxicity data (Kirkpatrick, 2005; Nemec, 2005; Kirkpatrick, 2008).
- Executive summary:
The potential of vaporized dimethyl disulfide (DMDS, CAS Reg. no. 624-92-0) to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow was assessed when administered via whole-body inhalation to Sprague Dawley rats for 6 hours per day for 3 consecutive days. The study was performed following the OECD Testing Guidelines 474 (29 July 2016).
In the range finding study, vaporized test substance (DMDS) was administered at target concentrations of 650 and 750 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2groups (Groups 2–3) ofCrl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. Afemale in the 750 ppm group was found dead immediately following exposure on Study Day 0. There were no clinical observations noted for the female found dead. There were no other test substance-related effects on survival. A single female in the 750 ppm group was observed with labored respiration on Study Day 2 prior to exposure. Yellow and clear material around the ventral neck and urogenital area were observed in the 650 ppm group males and females. In addition, a single 650 ppm group female was observed with vocalization during handling. There were no other test substance-related clinical observations. Test substance-related effects on body weight were noted in all test substance-exposed groups. Lower body weights were noted in the 650 and 750 ppm group males and females. Body weight losses or lower body weight gains were noted in the 650 ppm group males and females throughout the exposure period. These changes in body weight correlated with decreased food consumption in the test substance-exposed groups. Test substance-related effects on hematology was noted in all exposed group males and females. Higher red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and hemoglobin distribution width (HDW) and lower eosinophil (absolute) values were noted in all test substance-exposed groups. Lower neutrophil (percent and absolute) values and higher percent lymphocyte values were noted in the 650 and 750 ppm group males. Based on the microscopic findings noted in the 175 ppm group, it is possible that the effects on the white blood cells could be secondary to an inflammatory response in the nasal cavity at these higher concentrations. In addition, higher platelet values were noted in the 650 ppm group males and 750 ppm group males and females. Lower percent and absolute reticulocyte values were noted in the 750 ppm group males, but were not of sufficient magnitude to be indicative of bone marrow depression. Test substance-related effects in serum chemistry were noted in all exposed group males and females. Test substance-related higher albumin, total protein, and globulin values and lower alanine aminotransferase (ALT) were noted in all test substance-exposed group males and females. Higher creatinine and lower triglyceride values were noted in the 750 ppm group males. In addition, higher cholesterol values were noted in the 750 ppm group males and females. Test substance-related lower phosphorus values were noted in all test substance-exposed group males and was attributed to possible dehydration. Lower sodium dehydrogenase (SDH) was noted in the 650 ppm group males but was not attributed to the test substance as the change was not present in a dose-responsive manner. None of the effects on serum chemistry were considered to be evidence of dose-limiting toxicity that would have impacted the dose selection for Definitive Phase B. Test substance-related lower kidney and liver weights were noted in the 650 ppm group males and 750 ppm group males and females and higher lung weights relative to body weight were noted in 650 and 750 ppm group males and females. The differences were attributed to the lower final body weights in the test substance-exposed groups. The test substance was negative for bone marrow cytotoxicity in both male and female rats at 650 and 750 ppm. The concentration of 700 ppm was considered to be the maximal tolerated concentration for the definitive study.
In the definitive study, vaporized test substance (DMDS) was administered at target concentrations of 175, 350 and 700 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 3 groups (Groups 2–4) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. For Group 5, cyclophosphamide monohydrate (CP) was administered once daily on Study Days 0 and 1 (first and second days of exposure) orally by gavage. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1–4 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Group 5. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt (body weights only), on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Bone marrow was collected from 5 animals/sex/group from Groups 1–5 at the scheduled necropsy (between 2–4 hours following the last exposure or 18–24 hours following the last dose administration).All animals survived to the scheduled necropsy. There were no test substance-related clinical observations. Test substance-related lower mean body weight gains were noted in all test substance-exposed groups, which correlated with decreased food consumption in the DMDS-exposed groups. Dimethyl disulfide did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) of bone marrow compared to the vehicle control for male and female rats. In the absence of medullar toxicity, indirect evidence of the bone marrow exposure was provided by the systemic toxicity, including mortality and decrease of the body weight gain, observed in the range-finding and/or definitive studies and consistent with the available acute toxicity data (Kirkpatrick, 2005; Nemec, 2005; Kirkpatrick, 2008).
In conclusion, male and female Crl:CD(SD) rats were exposed to DMDS via whole-body inhalation for 6 hours per day for 3 consecutive days at exposure concentrations of 175, 350, and 700 ppm. Negative response for induction micronucleated polychromatic erythrocytes (%MN-PCEs) in bone marrow was obtained.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Study no WIL 795004: September 2016 to August 2017. Study no 00795005: May 2017 to September 2017. Both studies were performed at the request of an EU national competent authority in the frame of Regulation EC 1107/2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- A first comet assay was performed combined to a micronucleus assay (CRL report no. WIL-795004). In this study, the % Tail DNA for nasal tissue, liver, and lung from EMS-treated rats (both males and females) were often outside of the expected historical control ranges for each tissue. Therefore, all criteria for a valid assay were not met and it has been decided that the comet assay will be repeated (CRL report no. 00795005). Investigations were performed by Charles River and BioReliance Corporation to tentatively identify the origin of the failure (Appendices IV and V). Even if a definitive root cause was not determined, it does appear to be due to the electrophoresis chambers in use on study or the initial electrophoresis temperature. Both of these items have been addressed in the repeat comet study (CRL report no. 00795005) by replacement of the electrophoresis chambers used in this study with newly purchased chambers and a starting electrophoresis temperature of at least 4°C. Only the results from the range-finding assays of the first study (CRL report no. WIL-795004) are summarized in this RSS, the results of the comet assay are not presented since invalidated.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian comet assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 7-8 weeks old
- Weight at study initiation:
224 g to 293 g for males and from 149 g to 210 g for females in the Range-Finding Phase A groups
221 g to 263 g for males and from 155 g to 188 g for females in the Range-Finding Phase B groups
237 g to 295 g for males and from 156 g to 206 g for females in the definitive study
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 2 to 3 per cage by sex in clean, solid bottom cages
- Diet (ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water (ad libitum): reverse osmosis treated (on site) drinking water
- Acclimation period: at least 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.7–22.8
- Humidity (%): 44.2-56.2
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Vehicle:
- Air
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 500-L or 1000-L glass and stainless steel whole-body exposure chambers
- Method of holding animals in test chamber: individually in standard exposure batteries
- Source and rate of air: in-house compressed air
- Method of conditioning air: HEPA charcoal-filtered, temperature- and humidity-controlled supply air source
- System of generating vapors: Vapors of the test substance were
generated using a bubbler-type vaporization system. A 300-mL or 500-mL gas washing bottle, (Ace Glass, Inc.; Vineland, NJ) was filled with an appropriate amount of liquid test substance. Compressed nitrogen was metered into the inlet stem of the gas washing bottle and bubbled through a fritted disc. Nitrogen was metered to the gas washing bottle using a Coilhose regulator (Model No. 8802K) and controlled using a needle valve and Gilmont rotameter-type flowmeter. Concentrated vapors of the test substance were delivered to the exposure chamber inlet and diluted to the desired atmosphere concentration by mixing with the chamber supply air prior to entering the chamber.
- Temperature, humidity, pressure in air chamber: The mean temperature and mean relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively.
- Air flow rate:
Range-Finder Phase: 214-221 L/min Definitive Phase: 128-240 L/min
- Air change rate: at least 12 air changes per hour.
- Method of particle size determination: Microdust Pro 880nm aerosol monitoring system. Aerosol content was 0.0 mg/m3 for all high dose groups.
- Treatment of exhaust air: activated-carbon drum prior to passing through the facility exhaust system which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units
TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography/FIG
- Samples taken from breathing zone: yes - Duration of treatment / exposure:
- 6 hours per day
- Frequency of treatment:
- 3 consecutive days
- Post exposure period:
- 2 and 4 hours (DMDS exposed groups)
18–24 hours (positive control group) - Dose / conc.:
- 10 other: ppm (target)
- Remarks:
- 11 ppm (analytical) (group 2)
- Dose / conc.:
- 50 other: ppm (target)
- Remarks:
- 51 ppm (analytical) (group 3)
- Dose / conc.:
- 175 other: ppm (target)
- Remarks:
- 178 ppm (analytical) (group 4)
- Dose / conc.:
- 344 other: ppm (target)
- Remarks:
- 351 ppm (analytical) (group 5)
- Dose / conc.:
- 700 other: ppm (target)
- Remarks:
- 672 ppm (analytical) (group 6)
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, sham-exposed
- Positive control(s):
- Ethyl methanesulfonate (group 7)
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: oral, on study Days 1 and 2 (second and third day of exposure, respectively)
- Doses / concentrations: 200 mg/kg/day - Tissues and cell types examined:
- Nasal tissue, lung, and liver cells
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION (report no WIL-795004):
In order to assess the potential for exposure-limiting toxicity, the high target exposure concentration for the range-finding phases A and B (175 and 750 ppm, respectively) were selected based on the following previous inhalation studies. The mid- and low-target exposure concentrations for each phase were selected at intervals that were predicted to be narrow enough to reveal any concentration-r elated trends.
• Four-hour acute inhalation toxicity study in rats: LC50 was 1310 ppm. Mortality was 0/10, 4/10, 4/1 0, and 9/10 animals in the 847, 1188, 1308, and 1650 ppm groups respectively;
• Four-hour acute inhalation mammalian erythrocyte micronucleus assay in rats: no mortality up to 825 ppm. No significant reductions in the ratio of PCEs to total erythrocytes;
• Four-hour acute inhalation exposure in rats: increased serum glutamic-pyruvic-transaminase immediately after exposure to 600 ppm and increased gamma glutamyltransferase immediately after 500 ppm;
• Six-hour acute neurotoxicity study in rats: mortality was 0/24, 0/24 and 1/24 animals for the 100, 200, and 750 ppm group, respectively;
• Six-hour acute inhalation toxicity study in rats: no mortalities were observed up to 600 ppm. Acute inflammation and degeneration of the transitional and olfactory epithelia and acute inflammation of the respiratory epithelium were noted at 50, 150, 300, and 600 ppm, and degeneration of the respiratory epithelium was noted at concentration of 150 ppm and higher;
• Twenty-four-hour acute inhalation toxicity study: exposure-related degeneration of the olfactory epithelium at exposure levels of 9, 12.5, and 18 ppm and a slight increase in inflammation of the respiratory and olfactory epithelia at 18 ppm;
• Five-day subacute (6 hours/day) inhalation toxicity in rats:10 no mortalities were observed up to 600 ppm. Mean absolute lung and lung/body weights were higher in the 300 and 600 ppm group females. Hyperplasia of the squamous nasal epithelium was noted in = 300 ppm group males and all test substance-exposed group females (= 50 ppm). Hyperplasia of the transitional and respiratory epithelia and degeneration and regeneration of the olfactory epithelium were noted at all test substance exposure concentrations (= 50 ppm) in both sexes.
RANGE-FINDING STUDIES (report no WIL-795004)
In the range finding study A, vaporized test substance (DMDS) was administered at 10, 50 and 175 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2 groups (Groups 2–5) of Crl:CD(SD) rats (due to a technical error that resulted in the unscheduled deaths of animals in Group 4, administration of the 175 ppm exposure level was repeated (Group 5)). A concurrent control group (Group 1) received filtered air on a comparable regimen. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights and cage food weights were recorded within 4 days of receipt and on the day of randomization (body weights only), on the day the animals were placed into treatment groups (food weights only), and daily throughout the exposure period. The animals found dead or euthanized in extremis had a gross necropsy performed, nasal cavity with turbinates collected, and the carcasses were discarded without further tissue collection. All surviving animals were discarded without necropsy at the scheduled euthanasia. Samples of nasal tissue were collected from the surviving 3 animals/sex/group and were assessed for cell viability. The nasal cavity with turbinates from the remaining 3 animals/sex/group were collected for histopathologic examination.
In the range finding study B, vaporized test substance (DMDS) was administered at 650 anf 750 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2 groups (Groups 2–3) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights and cage food weights were recorded within 4 days of receipt and on the day of randomization (body weights only), on the day the animals were placed into treatment groups (food weights only), and daily throughout the exposure period. The animal found de ad had a gross necropsy performed and the carcass was discarded without tissue collection. Clinical pathology parameters (hematology and serum chemistry) were analyzed for all surviving animals within 3 hours following exposure on Study Day 2. Gross necropsies were conducted on all animals, and s elected organs were weighed at the scheduled necropsy. Selected tissues were collected possible future histopathology. Bone marrow was collected for evaluation of cytotoxicity from all surviving animals at the scheduled euthanasia (Study Day 2; between 2 and 4 hours following the last exposure).
TREATMENT AND SAMPLING TIMES:
All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1-6 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Group 7. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. At the time of necropsy on Day 2, nasal tissue from the 10, 50 and 175 ppm groups (2-4), liver and lung from the 175, 350 and 750 ppm groups (4-6) and all 3 organs from the negative (1) and positive control (7) groups were harvested at the Testing Facility for processing to single cell suspensions.
The head, lung, and liver were removed. Nasal tissue from the head was extracted. Prior to collection of the lung sample (for Groups 1, 4, 5, 6, and 7), the lung was lavaged to remove any potential inflammatory cells.
DETAILS OF SLIDE PREPARATION:
Quality Check Slides
Quality of the preparation of the comet slides was checked with control cells from Trevigen®. These control cells contain untreated (CC0), low (CC1), mid (CC2) and highly (CC3) damaged cell suspensions. These cells were shipped to Charles River Ashland from BioReliance on dry ice and stored in a freezer, set to maintain = 60ºC; prior to use.
Vials of control cells were thawed in warm water and retained on wet ice until centrifugation. Cells were then be centrifuged at 150 x g for 5 minutes at 2 to 8ºC after adding approximately 600 µL of ice cold Calcium and Magnesium free phosphate buffered saline (CMF-PBS). After centrifugation, approximately 600 µL of supernatant was removed. Vials of processed cell suspensions were retained on wet ice until Comet slides are prepared. A volume of 500 µL of 0.5% low melting point agarose was added to each vial containing approximately 50 µL of cell suspension to prepare Comet slides. Slides were prepared along with the assay slides and processed in same manner as the study slides. Each electrophoresis run included one control cell slide in the center position of the electrophoresis chamber. Each control cell slide had at least 4 wells/control cell concentrations including the untreated control.
Preparation of Slides
From each nasal, liver and lung suspension, an aliquot of 2.5 µL was mixed with 75 µL (0.5%) of low melting agarose. The cell/agrose suspension was applied to microscope slides commercially available pre-treated multi-well slides. Commercially purchased multi-well slides were used and these slides have 20 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify.
A portion of the remaining cell suspension (target volume ~500µL) was collected in labeled vial from each animal/organ and placed on dry ice. Prior to shipment, samples were stored in a freezer, set to maintain - 65ºC to -85°C. These vials were saved for possible future analysis. Following completion of the scoring of Comet slides, the results were reviewed by the Study Director and/or Principal Investigator for the Comet Assay and it was deemed that slides did not have to be prepared and scored from the frozen cells. Therefore, the frozen cell suspensions were discarded prior to report finalization.
Slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue, with the exception of the control cell slides. The control cell slides were labeled in a manner to indicate the electrophoresis run and the chamber information. At least two Trevigen, Inc 20-well slides were prepared per animal per tissue. Three slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.
Lysis
Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8°C.
Unwinding
After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl a minomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 4.0 – 6.5¿C and protected from light, allowing DNA to unwind.
Electrophoresis
Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 8.3 – 10.4°C and protected from light. The electrophoresis time was constant for all slides.
Neutralization
After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 2 hours and stored at room temperature with desiccant.
Cell Suspension and Slide Shipment
Slides of the processed nasal tissue, liver, and lung prepared by BioReliance at the Test Facility were stored at room temperature with desiccant. Cell suspensions were stored in a freezer, set to maintain 65ºC to 85°C. Once Comet slide preparation and electrophoresis were completed and the cells were fixed, the slides were shipped at ambient temperature to BioReliance for staining and scoring.
METHOD OF ANALYSIS:
Three slides/control cell suspension/electrophoresis run were used. Fifty randomly selected cell s were scored per slide, resulting in a total of 150 cells evaluated per control cell suspension/el ectrophoresis run. If one of the three slides does not have 50 scorable cells, additional cells were scored using the backup slides. If 150 cells are not available, then the calculations were performed using the number of scorable cells.
Three slides/wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV from Perceptive Instruments Ltd. (UK).
The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
• % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100; Olive et al. 1990)].
Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “ clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). Every effort was made to score at least 150 cells, otherwise, the total number of scorable cells was used for calculations. The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent (Collins et. al., 2004). “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.
CRITERIA FOR A VALID TEST
The group mean for the % tail DNA in the filtered air control group (negative control group) is expected to be within the historical vehicle control range. The positive control should induce responses that are compatible with those in the historical control and must be significantly increased relative to the concurrent filtered air control group (p = 0.05).
For the Trevigen® control cells in each electrophoresis run in each chamber; at least one dosed control cell concentration must have significant increase in % tail DNA compared to the untreated cell control.
EVALUATION OF TEST RESULTS
Once the criteria for a valid assay have been met, the results were evaluated as follows:
Means of 150 counts of % tail DNA, Tail moment, and Tail migration were presented for each animal and each organ. The mean and standard deviation of the mean values for % tail DNA were presented for each treatment group.
Statistical analysis was performed only for % tail DNA. If deemed necessary, other parameters of DN A damage (i.e. Tail moment) may be analyzed statistically and used in the overall assessment of DNA damage.
All conclusions were based on sound scientific judgment. As a guide to interpretation of the data, the following will be considered:
• The test substance will be considered to induce a positive response in a particular tissue if the mean % tail DNA (or other parameters of DNA damage) in one or more test substance groups (doses) is significantly elevated relative to the concurrent filtered air control group.
• The test substance will be judged negative for induction of DNA damage if no statistically significant increase in the mean % DNA damage (or other parameters) in the test substance groups relative to the concurrent filtered air control group is observed.
However, the results of the statistical analysis may not be the only criterion in determination of the test substance potential to induce DNA damage. The following may be taken in consideration:
• The historical vehicle control data; a statistically significant increase in the mean % DNA (or other parameters) may not be considered biologically relevant if the values do not exceed the range of historical vehicle control.
• Because cells undergoing necrosis or degeneration are prone to DNA degradation, independent of direct genotoxic effects of the test substance, doses that are found to be cytotoxic, by histopathology evaluation, may not be considered as relevant doses and may not be taken in consideration during the generation of the study conclusions. Accordingly, any statistically significant increase in DNA damage occurring at a cytotoxic dose may not be considered as a positive finding.
• A dose-dependent increase in the mean % tail DNA (or other parameters) across the dose levels tested; if a dose-response is evident with no statistically significant increase, additional testing, including histopathology evaluation of the tissue, may be considered.
• If criteria for either a positive or negative response are not met, the results may be judged as equivocal.
STATISTICS
The median %tail DNA for the Comets scored on each slide was determined and the mean of the median values was calculated for each animal. The mean of the individual animal was then used to calculate a group mean.
In order to quantify the test substance-related effects on DNA damage, the following statistical analysis will be performed:
• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test sub stance groups were compared using Levene’s test (significant level of p = 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p = 0.05).
• Linear regression analysis will be used to determine a dose response relationship (p < 0.01).
• A pair-wise comparison (Student’s T-test p<= 0.05) was used to compare the positive control group to the concurrent vehicle/negative control group.
Pair-wise comparison (Student’s t-test, p<= 0.05) were used to compare the data from the dosed control cell concentrations against the untreated control cell concentration. If needed, non-parametric statistical methods (Kruskal Wallis or Mann Whitney test) may be used in evaluation of data. This control cell analysis data will be included as part of the raw data package, but will not be included in the study report. - Statistics:
- The median %tail DNA for the Comets scored on each slide was determined and the mean of the median values was calculated for each animal. The mean of the individual animal was then used to calculate a group mean.
In order to quantify the test substance-related effects on DNA damage, the following statistical analysis will be performed:
• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p = 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p = 0.05).
• Linear regression analysis will be used to determine a dose response relationship (p < 0.01).
• A pair-wise comparison (Student’s T-test p<= 0.05) was used to compare the positive control group to the concurrent vehicle/negative control group.
Pair-wise comparison (Student’s t-test, p<= 0.05) were used to compare the data from the dosed control cell concentrations against the untreated control cell concentration. If needed, non-parametric statistical methods (Kruskal Wallis or Mann Whitney test) may be used in evaluation of data. This control cell analysis data will be included as part of the raw data package, but will not be included in the study report. - Sex:
- male/female
- Genotoxicity:
- negative
- Remarks:
- in liver, lung and nasal tissue
- Toxicity:
- yes
- Remarks:
- animals exposed up to the maximal tolerated concentrations
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY (report no. WIL-795004)
Range-Finding Phase A
On 03 Oct 2016 (Study Day 2), 11 of the 12 animals died during exposure and 1 of 12 females was euthanized in extremis following exposure to the test substance at a target exposure concentration of 175 ppm (Group 4). Due to an issue with the generation equipment, animals assigned to the 175 ppm group (Group 4) were exposed to a concentration above the reported LC50 value of the test substance on 03 Oct 2016, resulting in a repeat of the 175 ppm exposure (Group 5). All other animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on the viability of the nasal epithelium.
Test substance-related lower body weights were noted in the 50 and 175 ppm (Group 4) group males, which correlated with decreased food consumption. Test substance related decreased food consumption was also observed in the 175 ppm (Group 5) males.
Exposure to dimethyl disulfide (DMDS) for 3 days resulted in test substance-related alterations in 3 of 4 epithelial cell types at 50 and 175 ppm (Group 5) consistent with a direct irritant. Olfactory epithelium appeared to be the most sensitive epithelial cell population, based on incidence and severity of the findings, and the findings were most prominent at nasal levels III through V. Findings in the olfactory epithelium at nasal levels III, IV, and V showed a dose dependent increase in incidence and severity in both males and females. The incidence and severity of the findings in olfactory epithelium was also highest at nasal level IV. Neutrophil inflammation of transitional epithelium was typically associated with minimal ulceration of the transitional epithelium and considered a finding secondary to test substance administration.
Minimal to mild hyperplasia of the transitional epithelium was observed in males and females at 175 ppm while minimal hyperplasia was noted in males and females at 50 ppm and in 1 female in the 10 ppm group. Hyperplasia of the transitional epithelium has been well characterized as a common sequela to repeated exposures of inhaled xenobiotics and can regress after a recovery period of several weeks.
From all of the aforementioned test substance-related findings, only the minimal ulceration was considered adverse. The remaining findings were not considered to be adverse in the context of this study, as the area affected was limited and unlikely to have an effect on the function of the nasal mucosa.
Range-Finding Phase B
One female was found dead at 750 ppm immediately following exposure on Study Day 0. There were no clinical observations noted for the female found dead. There were no other test substance related effects on survival. A single female in the 750 ppm group was observed with labored respiration on Study Day 2 prior to exposure. Yellow material on the urogenital area and clear material on the ventral neck were observed in the 650 ppm group males and females. In addition, a single 650 ppm group female was observed with vocalization during handling on Study Day 0. There were no other test substance related clinical observations. Test substance-related effects on body weight were noted in all test substance-exposed groups. Significantly lower mean body weights were noted on Study Day 2 in the 650 and 750 ppm group males and females, with only the 650 ppm group females not being statistically significant. Statistically significant lower body weights were also noted in the 750 ppm group males on Study Day 1. Statistically significant mean body weight losses or lower mean body weight gains were noted in the 650 and 750 ppm group males and females throughout the entire exposure period. Test substance-related lower mean food consumption was noted in the 650 and 750 ppm group males and females throughout the exposure period. Test substance-related effects on hematology was noted in all exposed groups. Higher mean RBC, HGB, HCT, and HDW and lower mean absolute eosinophil values (statistically significant in all male groups) were noted in all test substance-exposed group males and females. Statistically significant lower mean neutrophil (percent and absolute) values and higher mean percent lymphocyte values were noted in the 650 and 750 ppm group males. In addition, significantly higher mean platelet values were noted in the 650 ppm group males and 750 ppm group males and females (statistically significant in both male groups). Statistically significant lower mean percent reticulocyte values were noted in the 750 ppm group males and nonstatistically significant lower mean absolute reticulocyte values were noted in the 750 ppm group males. None of the effects on hematology were considered to be evidence of dose-limiting toxicity that would have impacted the dose selection for Definitive Phase. Test substance-related effects on serum chemistry were noted in all exposed group males and females. Test substance-related higher mean albumin, total protein, and globulin values and lower ALT values were noted in all test-substance exposed group males and females. Higher mean creatinine (statistically significant) and lower mean triglyceride values were noted in the 750 ppm group males. In addition, statistically significant higher mean cholesterol values were noted in the 750 ppm group males and females. Test substance-related lower mean phosphorus values were noted in all test substance-exposed group males and was attributed to possible dehydration. Statistically significant low er mean SDH was noted in the 650 ppm group males but was not attributed to the test substance as the change was not present in a dose-responsive manner. None of the effects on serum chemistry were considered to be evidence of dose limiting toxicity that would have impacted the dose selection for Definitive Phase. The test substance did not produce a statistically significant decrease in the P CE:TE ratios at 650 and 750 ppm compared to the filtered air control for male and female rats. Test substance-related lower mean absolute liver weights were noted in the 650 ppm group males and 750 ppm group males and females, higher mean kidney weight relative to body weight was noted in the 750 ppm group females and higher mean relative lung weights were noted in 650 and 750 ppm group males and females. The differences were attributed to the lower final body weights in the test substance-exposed groups.
Based on the achieved exposure concentrations, the concentration of 700 ppm was selected as the high-dose in Definitive Phase, as exceeding this concentration would likely result in dose-limiting toxicity (mortality and/or moribundity).
RESULTS OF DEFINITIVE STUDY (report no. 795005)
Quality Check Slides
As % of Tail DNA in nasal tissue, liver and lung cells of the positive control animals were in the range of the expected values, the slides of the Trevigen® cells were not scores.
Clinical signs
All animals survived to the scheduled euthanasia.
Test substance-related thin body condition was noted for a single 700 ppm group male on Study Day 2. This observation corresponded with body weight loss during the same time period. Test substance-related lower mean body weights were noted in the 50, 175, 350, and 700 ppm group males and females. These lower body weights correlated with lower mean body weight gains and/or body weight losses. It should be noted that the body weight changes in the 175, 350, and 700 ppm group females were statistically significantly lower compared to the control group. However, the statistical significance is not reflected for mean body weights as the control group females were 2-9 grams lighter than the test substance-treated groups on Study Day 0. In addition, test substance-related lower mean food consumption was noted in the 50, 175, 350, and 700 ppm group males and in the 350 and 700 ppm group females. These differences correlated with the lower body weights in both sexes.
Comet assay Males
The % Tail DNA in nasal cells is summarized for each treatment group and presented in Table 1. Median values for the % Tail DNA, Tail moment and Tail migration (µm) for nasal cells are calculated per 150 cells for each animal and are presented in the Appendix I.
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 5.4%, which was lower than the % of clouds in the vehicle control group (13.0%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post- hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in nasal cells as compared to the vehicle control group (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the nasal (Appendix II).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.
The % Tail DNA in liver cells is summarized for each treatment group and presented in Table 2. Median values for the % Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal and are presented in the Appendix I.
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was between 0.6 and 1.2%, which was comparable with the % of clouds in the vehicle control group (1.2%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post- hoc analysis, was used in the statistical analysis of data.
• A statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance group (175 ppm) relative to the concurrent vehicle control group (ANOVA followed by Dunnett ’s post-hoc analysis, p < 0.05). However, this increase was not dose dependent and was within the historical range; therefore this increase was not considered biologically significant.
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control group (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver (Appendix II).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.
The % Tail DNA in lung cells is summarized for each treatment group and presented in Table 3. Median values for the % Tail DNA, Tail moment and Tail migration (µm) for lung cells are calculated per 150 cells for each animal and are presented in the Appendix I..
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was between 1.0 and 1.4%, which was comparable with the % of clouds in the vehicle control group (1.8%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post- hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the vehicle control group (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the lung (Appendix II).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.
Comet assay Females
The % Tail DNA in nasal cells is summarized for each treatment group and presented in Table 4. Median values for the % Tail DNA, Tail moment and Tail migration (µm) for nasal cells are calculated per 150 cells for each animal and are presented in the Appendix I.
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 16.6%, which was comparable with the % of clouds in the vehicle control group (16.2%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post- hoc analysis, was used in the statistical analysis of data.
• A statistically significant decrease in the % Tail DNA (DNA damage) was observed in the mid and high test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05).
• A dose-dependent decrease in the % Tail DNA was observed across three test substance doses (regression analysis, p < 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in nasal tis sue cells as compared to the vehicle control group (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the nasal ( Appendix II).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.
The % Tail DNA in liver cells is summarized for each treatment group and presented in Table 5. Median values for the % Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal and are presented in the Appendix I.
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the low and high dose test substance groups was 0.6 and 0.8% which was lower than with the vehicle control group and mid dose test substance group was 2.2%, respectively, which was comparable the vehicle control group (2.2%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• Statistically significant decrease in the % Tail DNA (DNA damage) was observed in the test substance group (700 ppm) relative to the concurrent vehicle control group (ANOVA followed by Dunnett ’s post-hoc analysis, p < 0.05).
• Dose-dependent decrease in the % Tail DNA was observed across three test substance doses (regression analysis, p < 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control group (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver (Appendix II).
• These results indicate that all criteria for a valid test, as specified in the protocol, were met.
• The % Tail DNA in lung cells is summarized for each treatment group and presented in Table 6. Median values for the % Tail DNA, Tail moment and Tail migration (µm) for lung cells are calculated per 150 cells for each animal and are presented in the Appendix I.
• The scoring results and a statistical analysis of data indicated the following:
The presence of ‘clouds’ in the test substance groups was = 1.4%, which was lower than% of clouds in the vehicle control group (2.6%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post- hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the vehicle control group (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the lung (Appendix II).
These results indicate that all criteria for a valid test, as specified in the protocol, were met. - Conclusions:
- Male and female Crl:CD(SD) rats were exposed to DMDS via whole-body inhalation for 6 hours per day for 3 consecutive days at exposure concentrations of 10, 50, 175, 350, and 700 ppm. Negative responses for DNA damage were obtained in nasal tissue at 10, 50 and 175 ppm and in liver and lung at 175, 350 and 700 ppm,
- Executive summary:
The potential of vaporized dimethyl disulfide (DMDS) to cause DNA damage in rats was assessed when administered via whole-body inhalation (Randazzo, 2017b). The in vivo alkaline comet was used for the detection of single and double stranded DNA breaks in cells or nuclei isolated from liver, lung, and nasal tissues. The study was performed following the OECD Testing Guideline 489 (29 July 2016).
To determine the concentrations to be used in the comet assay, a range-finding study was performed in 2 phases. The first one, as reported above in the summary of the micronucleus assay (Randazzo, 2017a), was performed to identify the maximal tolerated concentration for the comet assay in liver and lung. Seven hundreds (700) ppm was selected as the high-dose, as exceeding this concentration would likely result in dose-limiting toxicity (mortality and/or moribundity).
The second phase was performed to identify the maximal non cytotoxic concentration for the nasal tissue. Filtered air or DMDS was administered as a vapour to groups of 6 male and 6 female Crl:CD(SD) albino rats via whole-body exposure at concentrations of 0, 10, 50, or 175 ppm for 6 hours per day for 3 consecutive days. Surviving animals were sacrificed between 2 and 4 hours following the 3rd exposure. During this phase animals were observed for any signs of reaction to treatment. Individual body and food weights were recorded. Nasal turbinates were collected and examined for nasal cell viability and histopathologic examination. No other tissues were harvested. Due to technical error that resulted in the unscheduled death or sacrifice of all animals at 175 ppm, administration of the 175 ppm exposure level was repeated. All other animals survived to the scheduled necropsy. There were no DMDS-related clinical observations. Statistically significant lower mean body weight gains and cumulative body weight changes were noted in the 50 group males throughout the exposure period. Lower mean body weight gains were noted in the 175 ppm (repeated group) females, but did not reach statistical significance. Statistically significant higher body weights were noted in the 175 ppm males on Study Days 0 and 1due to the overall higher body weights of the 175 ppm animals in comparison to the filtered air control group animals. There were no effects on the viability of the nasal epithelium. Microscopic findings noted in nasal levels 2 and 3 of the 50 ppm groups included, but were not limited to, minimal degeneration of the translational epithelium (males), olfactory epithelium (males and females), and respiratory epithelium (females). Similar findings, often in increased incidence and/or severity were noted in the 175 ppm group males and females. These findings included minimal ulceration of the transitional epithelium, and minimal to mild degeneration of the transitional epithelium, olfactory epithelium, and respiratory epithelium. Due to the minimal to mild nature of the local effects, 175 ppm was selected as the high-dose since a target concentration higher than 175 ppm was expected to cause significant site-specific cytotoxicity that could interfere with the Comet assay.
In the main study, vaporized dimethyl disulfide (DMDS) was administered via whole-body inhalation for 6 hours per day for 3 consecutive days at 10, 50, 175, 350 and 700 ppm to 5 groups (Groups 2–6) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. Positive control, EMS (Group 7), was administered at 200 mg/kg/d on Study Days 1 and 2 (second and third day of exposure, respectively) orally by gavage. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1 6 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Group 7. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Samples of the nasal tissue from 5 animals/sex/group of groups 1-4 and 7, and samples of lung and liver from 5 animals/sex/group of groups 1 and 4-7 were collected at the Testing Facility for processing to single cell suspensions and mixing with 0.5% low melting agarose. The cell/agarose suspension was applied to commercially available pre-treated multi-well slides and retained at 2 - 8°C for at least 15 minutes to allow the gel to solidify. A retain sample of the remaining cell suspension was also stored frozen (-65ºC to -85°C) for possible future analysis. Following solidification of agarose, the slides were placed in jars containing commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8°C then washed with 0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5 and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water (pH was > 13). All slides remained in the buffer for 20 minutes at 2-10°C and protected from light, allowing DNA to unwind. Electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light. After completion of electrophoresis, the slides were removed and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 2 hours and stored at room temperature with desiccant prior to shipment to the principal investigator site (BioReliance) for slide staining with a DNA stain and scoring. The slides were identified with a random code. Three slides/wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using a fully validated automated scoring system.
All animals survived to the scheduled euthanasia. Test substance-related thin body condition was noted for a single 700 ppm group male on Study Day 2. This observation corresponded with body weight loss during the same time period. Test substance-related lower mean body weights were noted in the 50, 175, 350, and 700 ppm group males and females. These lower body weights correlated with lower mean body weight gains and/or body weight losses. It should be noted that the body weight changes in the 175, 350, and 700 ppm group females were statistically significantly lower compared to the control group. However, the statistical significance is not reflected for mean body weights as the control group females were 2-9 grams lighter than the test substance-treated groups on Study Day 0. In addition, test substance-related lower mean food consumption was noted in the 50, 175, 350, and 700 ppm group males and in the 350 and 700 ppm group females. These differences correlated with the lower body weights in both sexes.
There was no biologically significant increase in the % tail DNA in liver, lung, and nasal tissue cells of DMDS dosed male and female rats compared to the negative control. At 175 ppm in liver cells of male rats, a statistically significant increase was observed. Statistically significant decreases were also observed in the % tail DNA in Nasal Tissue and liver cells of DMDS dosed female rats. However, all these changes were within the range of the historical negative controls and did not bear any biological significance. Negative control values were within the expected range. Positive control values were compatible but sometimes slightly higher than the historical range.
Under the conditions of this study, the administration of DMDS did not cause a biologically significant increase in DNA damage in liver and lung at 175, 350, and 700 ppm, and in nasal tissue at 10, 50, and 175 ppm. Therefore, DMDS was concluded to be negative in the in vivo Comet Assay.
Referenceopen allclose all
Table1
Range-Finding Phase. Bone Marrow Data for Male Sprague Dawley Rats
Administered Dimethyl Disulfide for3 Consecutive Days (6 hrs/day)
TREATMENT |
ANIMAL No. |
PCEs |
NCEs |
PCE:TE Ratio |
|
Filtered Air |
1523 |
205 |
295 |
0.41 |
|
1524 |
281 |
219 |
0.56 |
||
1532 |
161 |
339 |
0.32 |
||
1539 |
234 |
266 |
0.47 |
||
1540 |
280 |
220 |
0.56 |
||
Mean ± SD |
|
|
0.46 ± 0.10 |
||
Dimethyl Disulfide |
1519 |
201 |
299 |
0.40 |
|
1529 |
284 |
216 |
0.57 |
||
1530 |
248 |
252 |
0.50 |
||
1535 |
254 |
246 |
0.51 |
||
1543 |
176 |
324 |
0.35 |
||
Mean ± SD |
|
|
0.47 ± 0.09 |
||
Dimethyl Disulfide |
1525 |
200 |
300 |
0.40 |
|
1526 |
181 |
319 |
0.36 |
||
1531 |
327 |
173 |
0.65 |
||
1541 |
199 |
301 |
0.40 |
||
1542 |
184 |
316 |
0.37 |
||
Mean ± SD |
|
|
|
0.44 ± 0.12 |
|
NCE = Normochromatic Erythrocyte |
|||||
TE = Total erythrocytes (PCE + NCE) |
PCE = Polychromatic Erythrocyte |
||||
Table2
Range-Finding Phase. Bone Marrow Data for Female Sprague Dawley Rats
Administered Dimethyl Disulfide for 3 Consecutive Days (6 hrs/day)
TREATMENT |
ANIMAL No. |
PCEs |
NCEs |
PCE:TE Ratio |
|
Filtered Air (Negative Control) |
1554 |
375 |
125 |
0.75 |
|
1556 |
206 |
294 |
0.41 |
||
1561 |
242 |
258 |
0.48 |
||
1564 |
263 |
237 |
0.53 |
||
1569 |
251 |
249 |
0.50 |
||
Mean ± SD |
|
|
0.53 ± 0.13 |
||
Dimethyl Disulfide |
1552 |
127 |
373 |
0.25 |
|
1558 |
286 |
214 |
0.57 |
||
1566 |
293 |
207 |
0.59 |
||
1568 |
197 |
303 |
0.39 |
||
1570 |
125 |
375 |
0.25 |
||
Mean ± SD |
|
|
0.41 ± 0.16 |
||
Dimethyl Disulfide |
1547 |
315 |
185 |
0.63 |
|
1555 |
268 |
232 |
0.54 |
||
1560 |
168 |
332 |
0.34 |
||
1563 |
230 |
270 |
0.46 |
||
Mean ± SD |
|
|
|
0.49 ± 0.12 |
|
NCE = Normochromatic Erythrocyte |
|||||
TE = Total erythrocytes (PCE + NCE) |
PCE = Polychromatic Erythrocyte |
||||
Table3
Definitive Phase. Micronucleus Assay Data for Male Sprague Dawley Rats
Administered Dimethyl Disulfide for 3 Consecutive Days (6 hrs/day)a
TREATMENT |
ANIMAL No. |
MN PCEs/ |
% MN-PCEs |
PCEs |
NCEs |
PCE:TE Ratio |
|
Filtered Air (Negative Control) |
1451 |
0 |
0.00 |
272 |
228 |
0.54 |
|
1459 |
3 |
0.08 |
241 |
259 |
0.48 |
||
1467 |
4 |
0.10 |
239 |
261 |
0.48 |
||
1475 |
0 |
0.00 |
299 |
201 |
0.60 |
||
1476 |
0 |
0.00 |
269 |
231 |
0.54 |
||
Mean ± SD |
|
|
0.04 ± 0.05 |
|
0.53 ± 0.05 |
||
Dimethyl Disulfide |
1445 |
6 |
0.15 |
190 |
310 |
0.38 |
|
1446 |
0 |
0.00 |
315 |
185 |
0.63 |
||
1450 |
2 |
0.05 |
255 |
245 |
0.51 |
||
1453 |
4 |
0.10 |
190 |
310 |
0.38 |
||
1463 |
0 |
0.00 |
256 |
244 |
0.51 |
||
Mean ± SD |
|
|
0.06 ± 0.07 |
|
0.48 ± 0.11 |
||
Dimethyl Disulfide |
1456 |
1 |
0.03 |
266 |
234 |
0.53 |
|
1460 |
0 |
0.00 |
202 |
298 |
0.40 |
||
1461 |
2 |
0.05 |
338 |
162 |
0.68 |
||
1473 |
6 |
0.15 |
144 |
356 |
0.29 |
||
1482 |
3 |
0.08 |
354 |
146 |
0.71 |
||
Mean ± SD |
|
|
0.06 ± 0.06 |
|
0.52 ± 0.18 |
||
Dimethyl Disulfide |
1448 |
4 |
0.10 |
151 |
349 |
0.30 |
|
1455 |
5 |
0.13 |
417 |
83 |
0.83 |
||
1457 |
3 |
0.08 |
247 |
253 |
0.49 |
||
1458 |
0 |
0.00 |
193 |
307 |
0.39 |
||
1478 |
12 |
0.30 |
114 |
386 |
0.23 |
||
Mean ± SD |
|
|
0.12 ± 0.11 |
|
|
0.45 ± 0.24 |
|
Cyclophosphamide (20 mg/kg/day)a |
1452 |
33 |
0.83 |
310 |
190 |
0.62 |
|
1468 |
64 |
1.60 |
186 |
314 |
0.37 |
||
1471 |
129 |
3.23 |
191 |
309 |
0.38 |
||
1472 |
120 |
3.00 |
86 |
414 |
0.17 |
||
1484 |
113 |
2.83 |
359 |
141 |
0.72 |
||
Mean ± SD |
|
|
2.30 ± 1.04* |
|
0.45 ± 0.22 |
||
MN = Micronucleated |
NCE = Normochromatic Erythrocyte |
||||||
TE = Total erythrocytes (PCE + NCE) |
PCE = Polychromatic Erythrocyte |
||||||
*Statistically different than negative controlp= 0.05. |
|||||||
aExcept for cyclophosphamide treatment; dosed for 2 consecutive days harvested approximately 18 to 24 hours after the second dose was administered |
|||||||
Table4
Definitive Phase. Micronucleus Assay Data for Female Sprague Dawley Rats
Administered Dimethyl Disulfide for 3 Consecutive Days (6 hrs/day)a
TREATMENT |
ANIMAL No. |
MN PCEs/ |
% MN-PCEs |
PCEs |
NCEs |
PCE:TE Ratio |
|
Filtered Air (Negative Control) |
1488 |
13 |
0.33 |
207 |
293 |
0.41 |
|
1489 |
11 |
0.28 |
267 |
233 |
0.53 |
||
1493 |
3 |
0.08 |
132 |
368 |
0.26 |
||
1498 |
15 |
0.38 |
304 |
196 |
0.61 |
||
1525 |
7 |
0.18 |
275 |
225 |
0.55 |
||
Mean ± SD |
|
|
0.25 ± 0.12 |
|
0.47 ± 0.14 |
||
Dimethyl Disulfide |
1499 |
8 |
0.20 |
220 |
280 |
0.44 |
|
1501 |
4 |
0.10 |
300 |
200 |
0.60 |
||
1504 |
6 |
0.15 |
138 |
362 |
0.28 |
||
1510 |
8 |
0.20 |
296 |
204 |
0.59 |
||
1513 |
12 |
0.30 |
333 |
167 |
0.67 |
||
Mean ± SD |
|
|
0.19 ± 0.07 |
|
0.51 ± 0.16 |
||
Dimethyl Disulfide |
1487 |
8 |
0.20 |
127 |
373 |
0.25 |
|
1496 |
4 |
0.10 |
243 |
257 |
0.49 |
||
1503 |
8 |
0.20 |
116 |
384 |
0.23 |
||
1507 |
2 |
0.05 |
281 |
219 |
0.56 |
||
1509 |
2 |
0.05 |
220 |
280 |
0.44 |
||
Mean ± SD |
|
|
0.12 ± 0.08 |
|
0.39 ± 0.15 |
||
Dimethyl Disulfide |
1490 |
12 |
0.30 |
322 |
178 |
0.64 |
|
1494 |
2 |
0.05 |
222 |
278 |
0.44 |
||
1500 |
1 |
0.03 |
302 |
198 |
0.60 |
||
1508 |
4 |
0.10 |
189 |
311 |
0.38 |
||
1516 |
9 |
0.23 |
185 |
315 |
0.37 |
||
Mean ± SD |
|
|
0.14 ± 0.12 |
|
|
0.49 ± 0.13 |
|
Cyclophosphamide (20 mg/kg/day)a |
1486 |
99 |
2.48 |
109 |
391 |
0.22 |
|
1505 |
38 |
0.95 |
133 |
367 |
0.27 |
||
1514 |
76 |
1.90 |
86 |
414 |
0.17 |
||
1517 |
91 |
2.28 |
197 |
303 |
0.39 |
||
1518 |
41 |
1.03 |
134 |
366 |
0.27 |
||
Mean ± SD |
|
|
1.73 ± 0.70* |
|
0.26 ± 0.08* |
||
MN = Micronucleated |
NCE = Normochromatic Erythrocyte |
||||||
TE = Total erythrocytes (PCE + NCE) |
PCE = Polychromatic Erythrocyte |
||||||
*Statistically different than vehicle controlp= 0.05. |
|||||||
aExcept for cyclophosphamide treatment; dosed for 2 consecutive days harvested approximately 18 to 24 hours after the second dose was administered |
|||||||
Table 1: % Tail DNA inNasal Cells Following Administrations in Male Rats
Samples collected 2 to 4 hours post-last dose |
|||||
|
|
|
|
|
|
Treatment |
Number of Animals |
Group Mean % of Clouds |
Tail DNA (%)A |
||
|
Mean |
± |
S.D. |
||
Vehicle Control:
Filtered Air |
5 |
13.0 |
0.57 |
± |
0.31 |
Test Article: Dimethyl Disulfide
10 ppm |
5 |
5.4 |
0.17 |
± |
0.15 |
50 ppm |
5 |
3.6 |
0.91 |
± |
0.61 |
175 ppm |
5 |
4.8 |
1.24 |
± |
1.41 |
Positive Control:
EMS 200 mg/kgB |
5 |
29.4 |
46.25 |
± |
8.25* |
AMean of 5 animals means of medians |
|||||
B Ethyl methanesulfonate (EMS), positive control for Comet assay, orally administered prior to organ collection on Study day 1 and 2. |
|||||
S.D. = Standard Deviation |
|||||
*p = 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control |
|||||
Table 2: % Tail DNA in Liver Cells Following Administrations in Male Rats
Samples collected 2 to 4 hours post-last dose |
|||||
|
|
|
|
|
|
Treatment |
Number of Animals |
Group Mean % of Clouds |
Tail DNA (%)A |
||
|
Mean |
± |
S.D. |
||
Vehicle Control: |
5 |
1.2 |
0.10 |
± |
0.04 |
Filtered Air |
|||||
Test Article: Dimethyl Disulfide |
5 |
0.6 |
0.33 |
± |
0.26# |
175 ppm |
|||||
350 ppm |
5 |
0.6 |
0.03 |
± |
0.01 |
700 ppm |
5 |
1.2 |
0.06 |
± |
0.02 |
Positive Control: |
5 |
6.6 |
48.44 |
± |
6.50* |
EMS 200 mg/kgB |
|||||
AMean of 5 animals means of medians |
|||||
S.D. = Standard Deviation |
|||||
*p = 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control |
|||||
# p = 0.05 (ANOVA, Dunnett’s post hoc); Statistically significant increase relative to the vehicle control |
|||||
Table 3: % Tail DNA in Lung Cells Following Administrations in Male Rats
Samples collected 2 to 4 hours post-last dose |
|||||
|
|
|
|
|
|
Treatment |
Number of Animals |
Group Mean % of Clouds |
Tail DNA (%)A |
||
|
Mean |
± |
S.D. |
||
Vehicle Control: |
5 |
1.8 |
0.11 |
± |
0.07 |
Filtered Air |
|||||
Test Article: Dimethyl Disulfide |
5 |
1.4 |
0.07 |
± |
0.02 |
175 ppm |
|||||
350 ppm |
5 |
1.2 |
0.06 |
± |
0.02 |
700 ppm |
5 |
1.0 |
0.08 |
± |
0.04 |
Positive Control: |
5 |
8.0 |
50.44 |
± |
9.80* |
EMS 200 mg/kgB |
|||||
AMean of 5 animals means of medians |
|||||
B Ethyl methanesulfonate (EMS), positive control for Comet assay, orally administered prior to organ collection on Study day 1 and 2. |
|||||
S.D. = Standard Deviation |
|||||
*p = 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control |
|||||
Table 4: % Tail DNA inNasalCells Following Administrations in Female Rats
Samples collected 2 to 4 hours post-last dose |
|||||
|
|
|
|
|
|
Treatment |
Number of Animals |
Group Mean % of Clouds |
Tail DNA (%)A |
||
|
Mean |
± |
S.D. |
||
Vehicle Control: |
5 |
16.2 |
1.31 |
± |
0.87 |
Filtered Air |
|||||
Test Article: Dimethyl Disulfide |
5 |
14.0 |
0.56 |
± |
0.38@ |
10 ppm |
|||||
50 ppm |
5 |
16.6 |
0.28 |
± |
0.19$@ |
175 ppm |
5 |
15.6 |
0.37 |
± |
0.36$@ |
Positive Control: |
5 |
40.6 |
34.81 |
± |
4.11* |
EMS 200 mg/kgB |
|||||
AMean of 5 animals means of medians |
|||||
S.D. = Standard Deviation |
|||||
*p = 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control |
|||||
@ p = 0.01 (regression analysis): Statistically significant relative to the vehicle control. |
|||||
$ p = 0.05 (ANOVA, Dunnett’s post hoc); Statistically significant decrease relative to the vehicle control. |
|||||
Table 5: % Tail DNA inLiverCellsFollowing Administrations in Female Rats
Samples collected 2 to 4 hours post-last dose |
|||||
|
|
|
|
|
|
Treatment |
Number of Animals |
Group Mean % of Clouds |
Tail DNA (%)A |
||
|
Mean |
± |
S.D. |
||
Vehicle Control: |
5 |
2.2 |
0.06 |
± |
0.03 |
Filtered Air |
|||||
Test Article: Dimethyl Disulfide |
5 |
0.6 |
0.04 |
± |
0.00@ |
175 ppm |
|||||
350 ppm |
5 |
2.2 |
0.04 |
± |
0.02@ |
700 ppm |
5 |
0.8 |
0.02 |
± |
0.01$@ |
Positive Control: |
5 |
3.8 |
34.99 |
± |
6.96* |
EMS 200 mg/kgB |
|||||
AMean of 5 animals means of medians |
|||||
S.D. = Standard Deviation |
|||||
*p = 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control |
|||||
@ p = 0.01 (regression analysis): Statistically significant relative to the vehicle control. |
|||||
$ p = 0.05 (ANOVA, Dunnett’s post hoc); Statistically significant decrease relative to the vehicle control. |
|||||
Table 6: % Tail DNA inLungCells Following Administrations in Female Rats
Samples collected 2 to 4 hours post-last dose |
|||||
|
|
|
|
|
|
Treatment |
Number of Animals |
Group Mean % of Clouds |
Tail DNA (%)A |
||
|
Mean |
± |
S.D. |
||
Vehicle Control: |
5 |
2.6 |
0.17 |
± |
0.06 |
Filtered Air |
|||||
Test Article: Dimethyl Disulfide |
5 |
0.6 |
0.12 |
± |
0.12 |
175 ppm |
|||||
350 ppm |
5 |
0.4 |
0.16 |
± |
0.07 |
700 ppm |
5 |
1.4 |
0.13 |
± |
0.08 |
Positive Control: |
5 |
5.4 |
33.96 |
± |
14.40* |
EMS 200 mg/kgB |
|||||
AMean of 5 animals means of medians |
|||||
B Ethyl methanesulfonate (EMS), positive control for Comet assay, orally administered prior to organ collection on Study day 1 and 2. |
|||||
S.D. = Standard Deviation |
|||||
*p = 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control |
|||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
IN VITRO STUDIES
In a key OECD 471 bacterial reverse mutation test (Wagner, 2007), dimethyl disulphide, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2uvrAin the presence and absence of Aroclor-induced rat liver S9. The assay was performed using the plate incorporation method. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate in the initial toxicity-mutation assay and 50, 150, 500, 1500 and 5000 µg per plate in the confirmatory mutagenicity assay. No positive mutagenic response was observed. Neither precipitate nor appreciable toxicity was observed. Dimethyl disulphide was concluded to be negative in the Bacterial Reverse Mutation Assay. In two other supporting OECD 471 bacterial reverse mutation tests, dimethyl disulphide was negative in Salmonella strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100, in the presence and absence of metabolic activation (Jones, 1985; Barfknecht, 1985).
In a key OECD 473 chromosome aberration study with human lymphocytes (De Vogel, 1990), dimethyl disulphide did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at non-toxic concentrations (<= 100 µg/mL), both in the absence and in the presence of metabolic activation. At the very toxic concentration of 300 µg/mL, both in the absence and in the presence of metabolic activation, dimethyl disulphide induced a statistically significant increase in the number of cells with structural chromosome aberrations.
In a key OECD 476 mammalian cell gene mutation assay (HGPRT) with CHO cells (Rutten, 1990), dimethyl disulphide (0.46, 1.37, 4.12, 12.3, 37.0, 74.0, 111, 333, 667 and 1000 µg/mL) did not increase the mutant frequency in the absence of metabolic activation. In the presence of a metabolic activation system, dimethyl disulphide induced a slight increase in mutant frequency at several concentrations. These increases were not concentration-related or clearly reproducible. Dimethyl disulphide is highly toxic to CHO cells at a concentration range from 74-1,000 µg/mL. The actual concentrations of dimethyl disulphide in culture medium were much lower than the target concentrations. Recovery experiments showed that about 50% was lost directly on incubation (presumably by evaporation) and during incubation an additional 25% is lost (presumably reactions with constituents of the incubation). There was no conclusive evidence for a genotoxic effect of dimethyl disulphide in cultured CHO cells.
In a key OECD 482 DNA damage and repair assay (Bichet, 1990), dimethyl disulphide (1, 5, 10, 50, 100, 200 and 300 µg/mL; cytotoxic>100 µg/mL) was not genotoxic to rat hepatocytes in culture.
IN VIVO STUDIES
In vivo comet assay (OECD 489)
The potential of vaporized dimethyl disulfide (DMDS) to cause DNA damage in rats was assessed when administered via whole-body inhalation (Randazzo, 2017b). The in vivo alkaline comet was used for the detection of single and double stranded DNA breaks in cells or nuclei isolated from liver, lung, and nasal tissues. The study was performed following the OECD Testing Guideline 489 (29 July 2016).
To determine the concentrations to be used in the comet assay, a range-finding study was performed in 2 phases. The first one, as reported above in the summary of the micronucleus assay (Randazzo, 2017a), was performed to identify the maximal tolerated concentration for the comet assay in liver and lung. Seven hundreds (700) ppm was selected as the high-dose, as exceeding this concentration would likely result in dose-limiting toxicity (mortality and/or moribundity).
The second phase was performed to identify the maximal non cytotoxic concentration for the nasal tissue. Filtered air or DMDS was administered as a vapour to groups of 6 male and 6 female Crl:CD(SD) albino rats via whole-body exposure at concentrations of 0, 10, 50, or 175 ppm for 6 hours per day for 3 consecutive days. Surviving animals were sacrificed between 2 and 4 hours following the 3rd exposure. During this phase animals were observed for any signs of reaction to treatment. Individual body and food weights were recorded. Nasal turbinates were collected and examined for nasal cell viability and histopathologic examination. No other tissues were harvested. Due to technical error that resulted in the unscheduled death or sacrifice of all animals at 175 ppm, administration of the 175 ppm exposure level was repeated. All other animals survived to the scheduled necropsy. There were no DMDS-related clinical observations. Statistically significant lower mean body weight gains and cumulative body weight changes were noted in the 50 group males throughout the exposure period. Lower mean body weight gains were noted in the 175 ppm (repeated group) females, but did not reach statistical significance. Statistically significant higher body weights were noted in the 175 ppm males on Study Days 0 and 1due to the overall higher body weights of the 175 ppm animals in comparison to the filtered air control group animals. There were no effects on the viability of the nasal epithelium. Microscopic findings noted in nasal levels 2 and 3 of the 50 ppm groups included, but were not limited to, minimal degeneration of the translational epithelium (males), olfactory epithelium (males and females), and respiratory epithelium (females). Similar findings, often in increased incidence and/or severity were noted in the 175 ppm group males and females. These findings included minimal ulceration of the transitional epithelium, and minimal to mild degeneration of the transitional epithelium, olfactory epithelium, and respiratory epithelium. Due to the minimal to mild nature of the local effects, 175 ppm was selected as the high-dose since a target concentration higher than 175 ppm was expected to cause significant site-specific cytotoxicity that could interfere with the Comet assay.
In the main study, vaporized dimethyl disulfide (DMDS) was administered via whole-body inhalation for 6 hours per day for 3 consecutive days at 10, 50, 175, 350 and 700 ppm to 5 groups (Groups 2–6) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. Positive control, EMS (Group 7), was administered at 200 mg/kg/d on Study Days 1 and 2 (second and third day of exposure, respectively) orally by gavage. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1 6 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Group 7. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Samples of the nasal tissue from 5 animals/sex/group of groups 1-4 and 7, and samples of lung and liver from 5 animals/sex/group of groups 1 and 4-7 were collected at the Testing Facility for processing to single cell suspensions and mixing with 0.5% low melting agarose. The cell/agarose suspension was applied to commercially available pre-treated multi-well slides and retained at 2 - 8°C for at least 15 minutes to allow the gel to solidify. A retain sample of the remaining cell suspension was also stored frozen (-65ºC to -85°C) for possible future analysis. Following solidification of agarose, the slides were placed in jars containing commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8°C then washed with 0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5 and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water (pH was > 13). All slides remained in the buffer for 20 minutes at 2-10°C and protected from light, allowing DNA to unwind. Electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light. After completion of electrophoresis, the slides were removed and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 2 hours and stored at room temperature with desiccant prior to shipment to the principal investigator site (BioReliance) for slide staining with a DNA stain and scoring. The slides were identified with a random code. Three slides/wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using a fully validated automated scoring system.
All animals survived to the scheduled euthanasia. Test substance-related thin body condition was noted for a single 700 ppm group male on Study Day 2. This observation corresponded with body weight loss during the same time period. Test substance-related lower mean body weights were noted in the 50, 175, 350, and 700 ppm group males and females. These lower body weights correlated with lower mean body weight gains and/or body weight losses. It should be noted that the body weight changes in the 175, 350, and 700 ppm group females were statistically significantly lower compared to the control group. However, the statistical significance is not reflected for mean body weights as the control group females were 2-9 grams lighter than the test substance-treated groups on Study Day 0. In addition, test substance-related lower mean food consumption was noted in the 50, 175, 350, and 700 ppm group males and in the 350 and 700 ppm group females. These differences correlated with the lower body weights in both sexes.
There was no biologically significant increase in the % tail DNA in liver, lung, and nasal tissue cells of DMDS dosed male and female rats compared to the negative control. At 175 ppm in liver cells of male rats, a statistically significant increase was observed. Statistically significant decreases were also observed in the % tail DNA in Nasal Tissue and liver cells of DMDS dosed female rats. However, all these changes were within the range of the historical negative controls and did not bear any biological significance. Negative control values were within the expected range. Positive control values were compatible but sometimes slightly higher than the historical range.
Under the conditions of this study, the administration of DMDS did not cause a biologically significant increase in DNA damage in liver and lung at 175, 350, and 700 ppm, and in nasal tissue at 10, 50, and 175 ppm. Therefore, DMDS was concluded to be negative in the in vivo Comet Assay.
Micronucleus assays (OECD 474)
In a key study, the potential of vaporized dimethyl disulfide (DMDS) to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow was assessed when administered via whole-body inhalation to Sprague Dawley rats for 6 hours per day for 3 consecutive days (Randazzo, 2017a). The study was performed following the OECD Testing Guidelines 474 (29 July 2016).
In the range finding study, vaporized test substance (DMDS) was administered at target concentrations of 650 and 750 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2 groups (Groups 2–3) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. A female in the 750 ppm group was found dead immediately following exposure on Study Day 0. There were no clinical observations noted for the female found dead. There were no other test substance-related effects on survival. A single female in the 750 ppm group was observed with labored respiration on Study Day 2 prior to exposure. Yellow and clear material around the ventral neck and urogenital area were observed in the 650 ppm group males and females. In addition, a single 650 ppm group female was observed with vocalization during handling. There were no other test substance-related clinical observations. Test substance-related effects on body weight were noted in all test substance-exposed groups. Lower body weights were noted in the 650 and 750 ppm group males and females. Body weight losses or lower body weight gains were noted in the 650 ppm group males and females throughout the exposure period. These changes in body weight correlated with decreased food consumption in the test substance-exposed groups. Test substance-related effects on hematology was noted in all exposed group males and females. Higher red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and hemoglobin distribution width (HDW) and lower eosinophil (absolute) values were noted in all test substance-exposed groups. Lower neutrophil (percent and absolute) values and higher percent lymphocyte values were noted in the 650 and 750 ppm group males. Based on the microscopic findings noted in the 175 ppm group, it is possible that the effects on the white blood cells could be secondary to an inflammatory response in the nasal cavity at these higher concentrations. In addition, higher platelet values were noted in the 650 ppm group males and 750 ppm group males and females. Lower percent and absolute reticulocyte values were noted in the 750 ppm group males, but were not of sufficient magnitude to be indicative of bone marrow depression. Test substance-related effects in serum chemistry were noted in all exposed group males and females. Test substance-related higher albumin, total protein, and globulin values and lower alanine aminotransferase (ALT) were noted in all test substance-exposed group males and females. Higher creatinine and lower triglyceride values were noted in the 750 ppm group males. In addition, higher cholesterol values were noted in the 750 ppm group males and females. Test substance-related lower phosphorus values were noted in all test substance-exposed group males and was attributed to possible dehydration. Lower sodium dehydrogenase (SDH) was noted in the 650 ppm group males but was not attributed to the test substance as the change was not present in a dose-responsive manner. None of the effects on serum chemistry were considered to be evidence of dose-limiting toxicity that would have impacted the dose selection for Definitive Phase B. Test substance-related lower kidney and liver weights were noted in the 650 ppm group males and 750 ppm group males and females and higher lung weights relative to body weight were noted in 650 and 750 ppm group males and females. The differences were attributed to the lower final body weights in the test substance-exposed groups. The test substance was negative for bone marrow cytotoxicity in both male and female rats at 650 and 750 ppm. The concentration of 700 ppm was considered to be the maximal tolerated concentration for the definitive study.
In the definitive study, vaporized test substance (DMDS) was administered at target concentrations of 175, 350 and 700 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 3 groups (Groups 2–4) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. For Group 5, cyclophosphamide monohydrate (CP) was administered once daily on Study Days 0 and 1 (first and second days of exposure) orally by gavage. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1–4 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Group 5. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt (body weights only), on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Bone marrow was collected from 5 animals/sex/group from Groups 1–5 at the scheduled necropsy (between 2–4 hours following the last exposure or 18–24 hours following the last dose administration).All animals survived to the scheduled necropsy. There were no test substance-related clinical observations. Test substance-related lower mean body weight gains were noted in all test substance-exposed groups, which correlated with decreased food consumption in the DMDS-exposed groups. Dimethyl disulfide did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) of bone marrow compared to the vehicle control for male and female rats. In the absence of medullar toxicity, indirect evidence of the bone marrow exposure was provided by the systemic toxicity, including mortality and decrease of the body weight gain, observed in the range-finding and/or definitive studies and consistent with the available acute toxicity data (Kirkpatrick, 2005; Nemec, 2005; Kirkpatrick, 2008).
In conclusion, male and female Crl:CD(SD) rats were exposed to DMDS via whole-body inhalation for 6 hours per day for 3 consecutive days at exposure concentrations of 175, 350, and 700 ppm. Negative response for induction micronucleated polychromatic erythrocytes (%MN-PCEs) in bone marrow was obtained
In a supporting micronucleus assay performed following the OECD guideline # 474 and the OPPTS Guideline # 870.5395 dimethyl disulphide at concentrations of 217, 421 and 825 ppm did not induce a statistically significant increase in the incidence of micronucleated polychromatic erythocytes in the bone marrow when male and female Sprague-Dawley rats were exposed to test article as a single, 4-hour, whole-body inhalation exposure (Weinberg, 2007).
In a supporting micronucleus assay, three groups of mice were exposed during 6 hours a day for 4 consecutive days to atmospheres containing 0, 250 and 500 ppm DMDS (Willems, 1989). Bone marrow cells were collected from the femur and examined for the presence of micronucleated poly- and normochromatic erythrocytes. Exposure to DMDS resulted in clear signs of toxicity at 250 ppm and 500 ppm, and 12 of the 20 mice of the 500 ppm group died. Mean numbers of polychromatic erythrocytes were slightly lower in mice exposed to 500 ppm DMDS, suggesting slight cytotoxic effects on bone marrow cells. There were no increases in the incidences of micronucleated erythrocytes attributable to DMDS exposure.
UDS assay (OECD 482)
In a key OECD 482 unscheduled DNA synthesis test (Rutten, 1990), male rats were exposed by inhalation for a period of 4 h to 500 ppm dimethyl disulphide(maximally tolerated concentration). Immediately after exposure and after subsequent non-exposure periods of 16 and 24 h, animals were sacrificed for the isolation of hepatocytes. The DNA-repair activities were examined by autoradiography in monolayer cultures of hepatocytes, incubated in the presence of [methyl-3H] thymidine. Dimethyl disulphide did not induce DNA-repair activities in hepatocytes, either during the 4 h exposure period or during the subsequent 16 h or 24 h after the exposure period.
Justification for classification or non-classification
Based on the battery of in vitro and in vivo genetic toxicology studies, DMDS is not a classified mutagen according to REGULATION (EC) No 1272-2008.
Justification of the non classification (RAC) :
Considering the negative results in all in vitro and in vivo tests at doses/ concentrations selected in line with OECD TG recommendations, RAC is of the opinion that DMDS does not warrant classification for germ cell mutagenicity.
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