Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
no guideline available
Principles of method if other than guideline:
For the Comet assay no internationally accepted guideline is available. This study was conducted according to the procedures indicated by the following recommendations: Tice, R.R. et al. (2000): Single Cell Gel/Comet Assay: Guidelines for ln Vitro and ln Vivo Genetic Toxicology Testing. Environmentaland Molecular Mutagenesis 35: 206-221 and Hartmann, A. et al. (2003): Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18 (1): 45-51
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Micronucleus Assay and Comet Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS:
- Strain: rat (Wistar HsdCpb: WU)
- Source: Charles River Laboratories, Research Models and Services Germany GmbH,Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 271.8 g (SD +/- 6.7 g)
- Housing: in groups in cage type Makrolon Type IV, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: It shows relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.
Details on exposure:
oral administration
Duration of treatment / exposure:
48h
Frequency of treatment:
All animals received three times (48h, 24h, 4h prior preparation) orally a single standard volume.
The animals of the positive control groups received the positive control substances once orally.
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
7 male rats/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
For the Micronucleus Assay: Cyclophosphamide (CPA)
- Administration: orally, once, 24h prior to preparation
- Doses / concentrations: CPA dissolved in sterile wate; 20 mg/kg b.w.
- Volume administered: 10 mL/kg b.w.

For the Comet Assay: Methylmethanesulphonate (MMS)
- Administration: orally, once, 4h prior to preparation
- Doses / concentrations: MMS dissolved in 0,9% NaCl solution; 25 mg/kg b.w.
- Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
Micronucleus Assay: bone marrow cells of tibias/femur
Comet Assay: hepatocytes and cells from the small intestine
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity of WS400402 was performed with at least two animals per sex and test group under identical conditions as in the mutagenicity study concerning the following parameters: animal strain; vehicle; route, frequency, and volume of administration. Since no gender-specific differences in toxicity were observed, the main experiment was performed using male animals only treated with doses of 200, 400, and 800 mg/kg b.w. WS400402, respectively.

TREATMENT AND SAMPLING TIMES: Samples were taken at 24h and 48h - covering the interval in which maximum frequencies of micronuclei occur. This could be too late for the assessment of the DNA strand breaks, as these might have undergone repair. For this purpose a shorter additional treatment interval has to be considered in order detect damaged DNA in the correct time frame. For those reasons the animals were treated three times with three doses of the test item at intervals of 48 h, 24 h and 4 h prior to preparation.

DETAILS OF SLIDE PREPARATION: Three slides per organ of the animals were prepared with 10 % cell suspension and 90 % 0.7 % (w/v) agarose (low melting point agarose). 100 µL was applied per slide. The slides were cooled before being submerged in lysis buffer.

METHOD OF ANALYSIS:
Micronucleus Assay: At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei by manual inspection. The micronucleated PCE´s per 2000 PCE´s and the ratio of polychromatic erythrocytes to the total number of erythrocytes (NCE + PCE) are presented for each animal.

Comet Assay: DNA strand breaks were analysed using the alkaline single cell gel electrophoresis. One hundred cells per animal were evaluated on coded slides with a fluorescence microscope and the damage of each nucleus was measured and recorded by an image analysis programme (Comet Assay IV, Perceptive Instruments).

Evaluation criteria:
Micronucleus Assay: A test item was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the tested dose levels.
Comet Assay: A test item was classified as mutagenic if it induced either a dose-related increase or a biologically relevant increase in the tail % intensity in a single dose group as compared to the historical control range.
Statistics:
Micronucleus Assay: This was confirmed by means of the nonparametric Mann-Whitney test.
Comet Assay: Normally distributed data was analysed using a one-tailed student´s t-test. Not normal distributed data were analysed using a Mann-Whitney rank sum test. However, the primary point of consideration was the biological relevance of the results.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Micronucleus assay in bone marrow cells
Toxicity:
no effects
Remarks:
no cytotoxicity observed in bone marrow cells
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Comet assay in liver cells
Toxicity:
no effects
Remarks:
no cytotoxicity observed in liver cells
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Comet assay in cells of small intestine
Toxicity:
yes
Remarks:
cytotoxicity observed in cells of small intestine at each dose level without showing a dose-response effect
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 800 and 1250 mg/kg b.w.
- Clinical signs of toxicity in test animals: Severe toxic symptoms after the second treatment at the high concentration; the moribund animals were sacrificed. Animals treated three times with 800 mg/kg b.w. showed clear signs of toxicity, e.g. hunchback, ruffled fur. This concentration was taken as the highest one in the definitve study.

RESULTS OF DEFINITIVE STUDY
- Micronucleus assay: There was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei after administration of the test material and with any dose level used. The observed values in all test material treated groups are below the concurrent solvent controls; no micronuclei induced at none of the tested concentrations. The results table is attached as background material.
- Comet assay:
The comet assay on cells of the liver did not reveal a biologically relevant or statistically significant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Tail Intensity). All obtained test material group values were below or near to the values of the vehicle control group and additionally within the historical vehicle control data range.

The comet assay on cells of the small intestine, i.e. the first site of contact of cells with the substance, did not reveal an increase in DNA damage up to the highest dose level although at all dose levels cytotoxicity was observed. The results tables are attached as background material. PLEASE NOTE: in the results tables there is a hand written correction of the claculated group mean value of high dose animals. This wrong value was reported in the final report and interpreted as DNA damage in intestinal cells. However, the correct group mean value shows that there was no DNA damage. Discussion with the laboratory to revise the report were started on 26 June 2019.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
This in vivo genotoxicity test was performed because in two in vitro tests positive results were obtained for clastogenicity and for gene mutation.
In the micronucleus test-part of this study, induction of micronuclei in bone marrow cells was not detected up to the highest dose.

For evaluation of an in vivo potential for gene mutation in liver cells the Comet assay was performed instead of a test for unscheduled DNA synthesis (UDS). The positive control substance induced genotoxicity in liver cells whereas with the test substance WS400402 no effects were observed up to the highest dose. From these results it can be stated that under the experimental conditions reported, WS400402, up to the maximum dose level tested, did neither induce micronuclei as determined by the micronucleus test in bone marrow cells nor primary DNA damage in the liver as determined by the Comet assay.

Next to these two endpoints in bone marrow cells and liver cells also cells of the small intestine, i.e. the first site of contact with the test substance, were investigated for DNA damage by the Comet assay. Whereas in bone marrow and liver cells no cytotoxicity was observed, in cells of the small intestine cytotoxicity was observed at each dose level. However, no DNA damage was observed up to the highest dose level.