Molybdenum, bis(N,N-dibutylcarbamodithioato-κS,κS')dioxodi-μ-thioxodi-, stereoisomer

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Method B62: In vivo Mammalian Alkaline Comet Assay

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29. July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Molybdenum, bis(N,N-dibutylcarbamodithioato-κS,κS')dioxodi-μ-thioxodi-, stereoisomer

EC Number:

825-571-0

Cas Number:

60428-79-7

Molecular formula:

C18H36Mo2N2O2S6

IUPAC Name:

Molybdenum, bis(N,N-dibutylcarbamodithioato-κS,κS')dioxodi-μ-thioxodi-, stereoisomer

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recommended test system

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 6 - 10 weeks (start of treatment)
- Weight at study initiation: 169 - 190 g
- Assigned to test groups randomly: yes
- Housing: group
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65 %
- Air changes (per hr): at least eight air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Propylene glycol
- Vehicle(s)/solvent(s) used: propylene glycol
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals and ability to form a suitable dosing formulation.
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.

Details on exposure:

The preparations were made freshly before each dosing occasion.
The test item was formulated in propylene glycol. Vortexing was used to formulate the test item.
The formulations were constantly stirred during application using a magnetic stirrer.

Duration of treatment / exposure:

Dosing 24 h and 4 h prior to cell preparation.

Frequency of treatment:

twice

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethanesulfonate

Examinations

Tissues and cell types examined:

Cells from the liver
Cells from the small intestine

Details of tissue and slide preparation:

Cells from the liver:

A piece of the liver (about 1 cm diameter) was cut off and rinsed several times in ice-cold mincing buffer and then minced in ice-cold mincing buffer using forceps. The resulting cell suspension was filtered through a 70 μm cell strainer. An adequate volume of the cell suspension was centrifuged at about 10000 g for 1 min and the resulting pellet suspended in prewarmed 0.7 % agarose.

Cells from the intestine:

A piece of the small intestine (duodenum/jejunum, approximately 5 cm distal from the pylorus) was cut off and rinsed several times in cold HBSS, excess fat was removed and a piece of about 1 cm length was rinsed several times in ice-cold mincing buffer and then minced in ice-cold mincing buffer using fine scissors. The resulting cell suspension was filtered through a 70 μm cell strainer. An adequate volume of the cell suspension was centrifuged at about 10000 g for 1 min and the resulting pellet suspended in prewarmed 0.7 % agarose.

Four slides per tissue and per animal were prepared with 10 % cell suspension and 90 % of a 0.7 % (w/v) agarose (low melting point agarose) solution. 100 μL were applied per slide. The slides were cooled before being submerged in lysis buffer.
The following steps of protocol were performed with the slides:
Lysis 1h up to 2 days in Lysis buffer pH 10
Alkaline treatment 20 min in electrophoresis buffer
Electrophoresis 30 min in electrophoresis buffer 300 mA, 25 V, performed in a cool and dark environment (2 – 8°C)
Neutralisation about 10 min in neutralisation buffer
Dehydration approx. 2 min in 99 % ethanol
After dehydration the slides were air-dried and stored protected from dust and light until evaluation.

Evaluation criteria:

The DNA of the cells was stained with the fluorescence dye ethidium bromide (20 μg/mL; 40 μL per slide), immediately before evaluation.
All animals per test group, 150 cells per animal, 50 cells per slide, were evaluated on coded slides with a fluorescence microscope using a 20 x objective. The damage of each nucleus was measured and recorded by a validated image analysis program (Comet Assay IV, Perceptive Instruments, ICCR-Roßdorf validation number: DE0084COMET2). One slide per animal was kept as a reserve for possible re-evaluation.

The following criteria were used for analysis of slides:
• Only clearly defined non-overlapping cells were scored
• Nuclei from dead/ apoptotic cells were not scored
• Cells with unusual staining artefacts were not scored
• All other normal cells, 150/animal, were scored where possible
• the number of nuclei from apoptotic or necrotic cells per 1500 total nuclei, 500 nuclei per slide, were determined separately
After evaluation, the cover-slips were removed and the slides were washed once for approx. 2 min in 99 % ethanol. The slides were air-dried and stored protected from dust and light.

The study is considered valid as the following criteria are met:
- At least five animals per group are available for analysis
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database
- The concurrent positive control produces a statistically significant and biologically relevant positive response, ideally compatible with those generated in the historical control database
- Adequate numbers of cells and doses have been analysed
- The criteria for the selection of the highest dose are consistent with those described in chapter 3.5.2 (maximum tolerated dose).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

See summary of results attached as background material.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item S-600 did not induce any biologically relevant DNA damage in the in vivo Comet assay.

Therefore, the test item S-600 is considered to be non-genotoxic for the liver and small intestine.