Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2018 to 13 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione
EC Number:
276-763-0
EC Name:
5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione
Cas Number:
72676-55-2
Molecular formula:
C4-H2-N4-S6
IUPAC Name:
5-[(5-sulfanylidene-4,5-dihydro-1,3,4-thiadiazol-2-yl)disulfanyl]-2,3-dihydro-1,3,4-thiadiazole-2-thione
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Sprague Dawley (Hsd:SD)
Details on species / strain selection:
Rats are used routinely in this test. This outbred strain maximises genetic heterogeneity, which tends to eliminate strain-specific responses.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Virus antibody-free (VAF) animals were acclimated for five days and were judged to be healthy prior to utilization in the study. The animals were 6 weeks of age at the start of the treatment.

Test animals and environmental conditions were as described in the OECD 489 guidelines.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle(s)/solvent(s) used: Arachis oil (peanut oil, USP)
- Justification for choice of solvent/vehicle: Arachis oil was chosen based upon existing information regarding the limited solubility of the test material in water at high concentrations, a 1983 acute oral toxicity study using corn oil as the vehicle, and the preference of the laboratory for arachis oil over corn oil for the 14-day range finding study and OECD 422 studies using this test material.
- Concentration of test material in vehicle: The test material was prepared in arachis oil/peanut oil USP at 125 mg/mL and 250 mg/L.
- Amount of vehicle (if gavage or dermal): Low and mid dose test substance formulations and the vehicle control substance were administered at a dose volume of 4 mL/kg/dose. The high dose test substance formulation was administered at dose volumes of 8 mL/kg/dose.
- Type and concentration of dispersant aid (if powder): Not applicable
- Lot/batch no. (if required): MKBS3571V
- Purity: USP grade
Details on exposure:
Preparation of Control Substances
Neat EMS was prepared in 0.9% sodium chloride for injection (saline). The dosing formulation was prepared at a concentration of 20 mg/mL just prior to use.

Preparation of Test Substance Dose Formulations
The test substance dose formulations were prepared fresh on each day of use.

Each concentration was prepared by calibrating a suitable size amber glass vial, containing an amount of test substance appropriate to the target batch size. Approximately 70% of the total volume of vehicle was added to the vial, and the mixture was stirred magnetically. Additional vehicle was added to achieve the final target volume, and the formulation was again stirred magnetically and sonicated until uniform.
Duration of treatment / exposure:
Dose Administration
Low and mid dose test substance formulations and the vehicle control substance were administered at a dose volume of 4 mL/kg/dose. The high dose test substance formulation and positive control substance formulation were administered at dose volumes of 8 and 10 mL/kg/dose, respectively.
Frequency of treatment:
All animals in Group 1 (dosed with the vehicle), and Groups 2 through 4 (dosed with the test substance) were dosed once per day on two consecutive days (Study Days 1 and 2). The second dose occurred approximately 21 hours after the first dose.

Animals in Group 5 (dosed with the positive control) were dosed once approximately 3 to 4 hours prior to organ collection on Study Day 2.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Dose range-finding study: 3 per sex per dose at 500, 1000 and 2000 mg/kg bodyweight.

Definitive study: 6 males per dose at 0, 500, 1000 and 2000 mg/kg bodyweight; 3 males for positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (EMS)

Examinations

Tissues and cell types examined:
Liver, stomach and duodenum were evaluated for DNA damage; a section of each tissue was preserved for possble histopathology evaluation.
Details of tissue and slide preparation:
Sections of the liver, glandular stomach and duodenum were placed in chilled mincing solution and minced or scraped to release the cells. Cell suspensions were strained into pre-labeled tubes and kept on wet ice during preparation of the slides. An aliquot of the suspension was used to prepare the Comet slides.

From each suspension, an aliquot of cells was mixed with low melting agarose (0.5%) and the cell/agarose suspension was applied to commercially available pre-treated multi-well microscope slides. The slides were kept at 2 to 8°C for at least 15 minutes to allow the gel to solidify. At least two 20-well slides were prepared per animal per tissue and identified with a random code. Following solidification of the agarose, the slides were placed in jars containing lysis solution and were stored overnight at 2 to 8°C.

After lysis, slides/wells were washed with neutralization buffer and placed in the electrophoresis chamber. The chamber reservoirs were filled with alkaline buffer (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water (pH > 13)), and remained in the buffer for 20 minutes at 2-10°C, protected from light. Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light.

The slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant. Slides were stained with a DNA stain prior to scoring.
Evaluation criteria:
Fifty randomly selected, non-overlapping cells per slide/well were assessed and measured for Comet Tail Migration, % Tail DNA, and Tail Moment in a total of 150 cells per animal; if 150 cells were not available, then the calculations were performed using the number of scorable cells.
Each slide was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was also determined when possible. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.
Statistics:
The mean values of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA.

Since the differences and variations between groups were found not to be significant for liver and stomach, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). Levene’s test indicated heterogeneous group variances for duodenum (p = 0.05); therefore, the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, statistical analysis was performed using the parametric tests described above. A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (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 control group.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test substance gave a negative (non-DNA damaging) response in % Tail DNA for liver, duodenum and stomach of male rats. None of the test substance-treated animal slides had significant increases in % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, duodenum and stomach.

Applicant's summary and conclusion

Conclusions:
Administration of the test material at doses up to and including a dose of 2000 mg/kg/dose did not cause a significant increase in DNA damage in the liver, duodenum or stomach of male rats relative to the concurrent vehicle control. Therefore, the test material was concluded to be negative in the in vivo Comet Assay.
Executive summary:

The test substance was evaluated for its genotoxic potential using the Comet assay to assess induction of DNA damage in liver, stomach and duodenum cells of male rats. The study was performed to the standardized guideline OECD 489 under GLP conditions.

 

Arachis oil (peanut oil, USP) was selected as the vehicle. Low and mid test substance formulations and vehicle control substance formulation were administered at a dose volume of 4 mL/kg/dose. The high test substance formulation and positive control substance formulation were administered at dose volumes of 8 and 10 mL/kg/dose, respectively. All formulations were administered by oral gavage.

 

In the dose range-finding assay (DRF), the dose levels tested were 500, 1000 and 2000 mg/kg/dose in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/dose, which is the highest guideline recommended dose for this assay. The definitive assay dose levels tested were 500, 1000 and 2000 mg/kg/dose, administered by oral gavage once daily for two consecutive days approximately 21 hours apart.

 

The test substance gave a negative (non-DNA damaging) response in % Tail DNA for liver, duodenum and stomach of male rats. None of the test substance-treated animal slides had significant increases in % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, duodenum and stomach.

 

Under the conditions of this study, the administration of the test material at doses up to and including a dose of 2000 mg/kg/dose did not cause a significant increase in DNA damage in the liver, duodenum or stomach of male rats relative to the concurrent vehicle control. Therefore, the test material was concluded to be negative in the in vivo Comet Assay.