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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the Ames test no genetic toxicity was observed. In the in vitro micronucleus test in human lymphocytes the substance induced the formation of micronuclei in the absence and presence of S-9 mix.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : commercial (Trinova Biochem GmbH, Germany) from Sprague Dawley rats intraperitonelly dosed with Aroclor 1254
- method of preparation of S9 mix : S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium :
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) : Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively

Test concentrations with justification for top dose:

5.4, 17, 52, 164, 512, 1600 µg/plate in the absence and presence of S9 mix. Precipitation occurred at levels >= 164 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: suitable vehicle, also used as negative control

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation) : yes preincubation : yes

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 48 h
- Harvest time after the end of treatment (sampling/recovery times):


METHODS FOR MEASUREMENTS OF GENOTOXICIY

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment

Evaluation criteria:

The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

In the first experiment at 1600 µg/plate a decrease in the number of revertants was observed in absence of S-9

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity : no
- Individual plate counts : see tables of first and second experiments attached in "Attached background material"
- Mean number of revertant colonies per plate and standard deviation : see tables of first and second experiments attached in "Attached background material"

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells
o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells)

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Remarks on result:

other: negative

Conclusions:

no mutagenicity observed in this Ames test

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus 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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors: non-smoking volunteers, aged 30 years
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: unknown
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.0°C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium : S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP; 4 µmol HEPES.
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v)

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) : positive control with cyclophosphamide

Test concentrations with justification for top dose:

31, 63, 125 µg/ml at 3 h exposure +/- S9 mix
7.8, 16, 31, 63, 125, 250 µg/ml at 24 h exposure - S9 mix
The test substance precipitated at >= 125 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: suitable solvent, also used as negative control

- Justification for percentage of solvent in the final culture medium:

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 3 h exposure + and - S9 mix; 24 h in absence of S9 mix but in the presence of cytoB
- Harvest time after the end of treatment (sampling/recovery times): after the 3 h exposure a 24 h incubation followed in the presence of cytoB before fixation; cells exposed for 24 h were directly fixed.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. : cytoB was used as indicated in TREATMENT AND HARVEST SCHEDULE
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 replicate cultures; At least 1000 binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 mononucleated cells per culture were scored for micronuclei separately.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The following criteria for scoring of binucleated cells were used :
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were used:
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:


METHODS FOR MEASUREMENTS OF GENOTOXICIY

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see Tables attached

Remarks on result:

other: positive in this test

Conclusions:

The substance induced the formation of micronuclei in human lymphocytes and may be considered a clastogenic compound.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In the in vivo micronucleus test in bone marrow cells of mice no clastogenic or aneugenic activity was observed.

In the in vivo Comet assay in the rat, the test item did not induce any biologically relevant DNA damage in the cells of the liver and small intestine.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

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

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

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

Dose / conc.:

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

Dose / conc.:

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

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

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).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

See summary of results attached as background material.

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 30-38 g
- Assigned to test groups randomly: yes
- Fasting period before study: no, feed was withheld 4 hours prior to dosing
- Housing: group housing (max 5 animals)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 42-61
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: propylene glycol;
- Justification for choice of solvent/vehicle: suitable vehicle
- Concentration of test material in vehicle: 100, 200, 400 mg/ml
- Amount of vehicle (if gavage or dermal): 5 ml/kg body weight
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity:

Details on exposure:

Dosing was split to two doses on the same day with 1 - 2 hour interval

Duration of treatment / exposure:

2 days

Frequency of treatment:

once daily

Post exposure period:

sampling time was 48 hours after the first dosing

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): suitable positive control
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg body weight

Tissues and cell types examined:

Bone marrow of the femurs was prepared and erythrocytes were examined

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: the maximum dose of 2000 mg/kg body weight was well tolerated by males and females in a pre-test; in the main study only males were used.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): animals were dosed twice with a 24 hour interval; samples were prepared 48 hours after the first dose.

DETAILS OF SLIDE PREPARATION: A drop of the bone marrow cell suspension in fetal calf serum was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated cover slipper.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: the limit dose of 2000 mg/kg body weight was well tolerated in males and females
- Solubility: suspension in propylene glycol
- Clinical signs of toxicity in test animals: no signs observed
- Evidence of cytotoxicity in tissue analyzed: not analysed
- Rationale for exposure: not applicable
- Harvest times: not applicable
- High dose with and without activation: not applicable
- Other:

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei observed
- Ratio of PCE/NCE (for Micronucleus assay): unchanged in dosed animals (see Table on PCE/NCE ratio attached as background material)
- Appropriateness of dose levels and route: dosing was up to the limit dose of 2000 mg/kg
- Statistical evaluation: not applicable

Conclusions:

The test substance S-600 was not clastogenic or aneugenic in the bone marrow micronucleus test up to the highest dose of 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In the Ames test no genotoxicity was observed in the absence or presence of S-9 mix. In the in vitro micronucleus test in human lymphocytes the substance induced the formation of micronuclei in the absence and presence of S-9 mix. In the in vivo micronucleus test in bone marrow cells of mice no clastogenic or aneugenic activity was observed. In the in vivo Comet assay in the rat, the test item did not induce any biologically relevant DNA damage in the cells of the liver and small intestine. Therefore, it is concluded that the substance S-600 is not genotoxic in vivo and accordingly, the substance does not need to be classified according to Regulation 1272/2008.