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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2012 to 31 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Official notice of MHLW, METI and MOE (31 March 2011) YAKUSHOKUHATSU 0331 No 7 SEIKYOKU No 5 KANPOKIHATSU No 110331009.
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Rikabinol HB
- Substance type: Monomer
- Physical state: White solid
- Analytical purity: 95.6%
- Lot/batch No.: 7095
- Expiration date of the lot/batch: 31 December 2012
- Storage condition of test material: Room temperature in the dark
- Other: Date received 12 December 2011

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
CELLS USED
Human blood was collected aseptically from two healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a 5% CO2 atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.
Metabolic activation:
with and without
Metabolic activation system:
S9 mixType and composition of metabolic activation system:
- source of S9
Preparation of S9 fraction
The S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver. The S9 fraction was purchased from a commercial source and stored at -80°C or below.

Preparation of S9 mix
S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilised before use.
Test concentrations with justification for top dose:
100, 150, 200, 250, 300, 350, 400, 450 and 500 µg/mL.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: solubility
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9 mix.
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 2 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 3 hours
- Selection time (if incubation with a selection agent): 10min

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Other: Metaphase analysis
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: positive with S9 fraction present

Applicant's summary and conclusion

Conclusions:
It is concluded that Rikabinol HB has shown evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system in the presence of S9 mix only, under the experimental conditions described. The test substance Rikabinol HB has also shown statistically significant increases in numerical aberrations in the form of polyploidy in this in vitro cytogenetic test system, under the conditions described.