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Administrative data

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

Data source

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

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
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:
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:

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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

Applicant's summary and conclusion

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.

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