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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 October 2014 to 14 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Micronucleus assay, bone marrow cells

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
Lot/Batch #: COD-002002
Description: Yellowish solid
Purity: 95.7%
Stability of test compound: Stability in solvent confirmed

Test animals

Species:
mouse
Strain:
NMRI
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: About 6 - 10 weeks
- Weight at study initiation: About 38.5 g
- Assigned to test groups randomly: Yes
- Housing: The mice were singly housed in Makrolon Type II/III with wire mesh tops and granulated soft wood bedding.
- Diet: Pelleted standard diet (certified), ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45% - 65%
- Photoperiod (hrs dark / hrs light): 12-hour light-dark cycle (on at 6 am)

IN-LIFE DATES: From 5-Oct-2014 to 14-Nov-2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Aqueous 0.5% methyl cellulose
- Justification for choice of vehicle: 0.5 % methyl cellulose was selected in order to compare the results with previous toxicity data; it is also a common vehicle in toxicity studies.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Ultraturrax treatment was used to formulate the test item. A correction factor of 1.04 was applied to the formulations.

Dose volume applied: 10 mL/kg bodyweight

Preliminary range finding test:
Mice (2 per/sex/dose group) were orally treated once at 1000, 1500 and 2000 mg/kg bw; the animals were observed for clinical signs of toxicity at intervals of approx. 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration

Duration of treatment / exposure:
24 hours
48 hours (additional control and high dose groups)
Frequency of treatment:
once
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7
Control animals:
yes
Positive control(s):
Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 40 mg/kg bodyweight

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCEs)
Normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the Preliminary Range Finding Test.

TREATMENT AND SAMPLING TIMES
Sampling of the bone marrow cells from all test substance groups, negative and positive control groups were conducted 24 hours after administration. Additional negative control and high dose animals were allocated to a 48 hours sampling time point.
To confirm exposure, additional satellite animals were allocated to the study and blood samples (0.5 mL) were taken from 3 males/per time point as follows: 1 and 4 hours for the high dose group and 1 hour for the negative control group.

DETAILS OF SLIDE PREPARATION
The cells prepared earlier were re-suspended in a small residual volume of serum and a small drop of the cell suspension was smeared onto a glass slide. At least one slide was prepared from each animal. The smear slides were thoroughly air-dried and then stained with May-Grünwald/Giemsa; coverslips were mounted with EUKITT. Slides were coded for identification.

METHOD OF ANALYSIS
Slides were examined for each animal under NIKON microspores with 100x oil immersion objectives. Per animal 4000 polychromatic erythrocytes (PCE) were analyzed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed as the ratio of polychromatic to total erythrocytes.
Evaluation criteria:
Definitions of micronuclei stained with Giemsa were as follows: Micronuclei exist in an erythrocyte; the diameter of micronuclei is half or less than mature erythrocytes; color of the micronuclei is same as the nuclei of adjacent leukocytes. Criteria for distinguishing polychromatic erythrocytes from normochromatic ones in smears stained with Giemsa were taken as follows. Polychromatic erythrocytes are blue-violet in color and normochromatic erythrocytes are grayish pale red in color. Therefore erythrocytes that were apparently red in color in a field of the view were classified as normochromatic erythrocytes and all the other erythrocytes were scored as polychromatic erythrocytes.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test, except for the 48 h high dose group for which significance was evaluated by means of the ANOVA.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: There was a single mortality at 1500 and 2000 mg/kg bw, but no mortality at 1000 mg/kg bw. Clinical signs included dyspnea, hunched posture, abdominal posture, tumbling, reduced activity and ruffled fur; there was no notable difference in the response between males and females. The presence of clinical signs confirms the systemic availability of the test material.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Males given 1000 mg/kg bw had reduced activity for one hour after treatment and ruffled fur for up to 48 hours after treatment. There were no clinical signs of toxicity at 500 or 250 mg/kg bw.
- Induction of micronuclei: There were no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes at any dose level. In the positive control group dosed with cyclophosphamide, the frequency of micronucleated polychromatic erythrocytes showed a highly significant increase.
- Ratio of PCE/NCE: 54- 57; The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item did not have any cytotoxic properties in the bone marrow. There was a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes in the positive control group.

Applicant's summary and conclusion