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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

HBPA showed no evidence of mutagenic activity in an in vitro gene mutation study in bacteria under the test conditions employed.

HBPA has shown evidence of causing an increase in the frequency of structural chromosome aberrations in an in vitro cytogenetic test system in the presence of S9 mix only, under the experimental conditions described. HBPA 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.

HBPA did not demonstrate mutagenic potential in this in vitro cell mutation assay.

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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
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
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
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. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
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
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
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
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.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2012 to 09 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y mouse lymphoma (3.7.2c) cells, were obtained from American Type Culture Collection (ATCC), Virginia. These cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.
Additional strain / cell type characteristics:
other: Heterozygous at the thymidine kinase locus (TK+/-)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C.

Preparation of S9 mix
S9 mix contains: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before adding to S9 fraction and R0.
Test concentrations with justification for top dose:
Preliminary toxic test; 4.7, 9.39, 18.78, 37.56, 75.13,150.25, 300.5, 601, 1202 and 2404 μg/mL
Mutation tests; 50, 75, 150, 175, 200, 225, 250, 275, 300 and 600 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
In the absence of S9 mix.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
In the presence of S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: Preliminary toxicity test; Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix
and for 24 hours in the absence of S9 mix.
Main mutation test; 3 hour exposure in the absence and presence of S9 mix and 24 hour exposure in the absence of S9 mix
- Selection time (if incubation with a selection agent): After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates.
- Fixation time (start of exposure up to fixation or harvest of cells): 3 and 24 hours.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1E10+06 to 1E10+07

DETERMINATION OF CYTOTOXICITY
- Method: Mutant frequency and cloning efficiency

Evaluation criteria:
Criteria for assessing mutagenic potential

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:

Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6 (Moore etal., 2006). The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:

If the linear trend test was negative, the result was regarded as negative.

If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s). Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
It was concluded that Rikabinol HB did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2012 to 28 May 2012
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
The study targets genes which are normally capable of synthesising the essential amino acid, histidine in the case of S. typhimurium and tryptophan in the case of Escherichia coli but the mutant strains used in this test are incapable of this function.

When these strains are exposed to a mutagen, reverse mutation to the original histidine / tryptophan-independent form takes place in a proportion of the population.

The strains used carry additional mutations that render them more sensitive to mutagens. The S. typhimurium strains have a defective cell coat, which allows greater permeability of test substances into the cell. All the strains are deficient in normal DNA repair processes. In addition, three of them possess a plasmid (pKM101) which introduces an error-prone repair process, resulting in increased sensitivity to some mutagens.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S-9 mixType and composition of metabolic activation system:
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80°C.

Preparation of S9 mix
The 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), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg/ plate.
The highest concentration tested was 50 mg/mL (5000 μg/plate), is the standard limit concentration recommended in the regulatory guidelines.
Vehicle / solvent:
- Vehicle; DMSO;
- Justification for choice of solvent/vehicle: The Sponsor indicated that Rikabinol HB was insoluble in water. Its solubility was determined at 50 mg/mL in DMSO and was found to be soluble. DMSO (ACS spectrophotometric grade) was, therefore, used as the vehicle for this study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
DURATION
1st Test - The plate incorporation assay
- Preincubation period: n/a
- Exposure duration: 72h at 37°C

2nd Test - The pre-incubation assay
- Preincubation period: 30 min at 37°C
- Exposure duration: 72h at 37°C

NUMBER OF REPLICATIONS: 3



Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None
Conclusions:
It was concluded that Rikabinol HB showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

A Bacterial reverse mutation test was conducted (Huntingdon Life Sciences, 2012, Study MOG0010) to examine the potential for HBPA to cause gene mutation. The study was conducted according to EC Method B.13/14, OECD test guideline 471, EPA test guideline OPPTS 870.5100 and Japan Guidelines for Screening Mutagenicity Testing Of Chemicals, and in compliance with GLP.

The strains of Salmonella Typhimurium (TA1535, TA1537, TA98, and TA100) and Escherichia Coli WP2 uvrA (pKM101) were exposed to HBPA as a solution in Dimethylsulphoxide (DMSO) using a standard plate incorporation assay and the pre-incubation methods at seven dose levels, in triplicate in both the presence and absence of metabolic activation. The six concentrations of HBPA were 5, 15, 50, 150, 500, 1500 and 5000 µg/ plate.

Toxicity (as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was seen in all strains following exposure to HBPA at 5000 μg/plate in both tests. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to HBPA at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

It was concluded that HBPA showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

 

 

A Chromosome Aberration Test in Human Lymphocytes was conducted (Huntingdon Life Sciences, 2012, Study MOG0011) do determine the potential for HBPA to cause chromosomal aberrations in Human Lymphocytes cultured in vitro. The study was conducted according to EU Method B.10, OECD test guideline 473, EPA test guideline OPPTS 870.5375 and Japan MHLW, METI and MOE, and in compliance with GLP.

The cultures were treated with HBPA for 3 hours in the presence and absence of metabolic activation followed by 18 hours recovery.The following concentrations were selected for metaphase analysis: in the absence of S9 mix: 250, 300 and 350 μg/mL; in the presence of S9 mix: 400, 475 and 500 μg/mL.

In the absence of S9 mix HBPA caused a reduction in the mitotic index to 49% of the vehicle control value at 350 μg/mL. In the presence of S9 mix HBPA caused a reduction in the mitotic index to 52% of the vehicle control value at 500 μg/mL.

 

In the absence of S9 mix, HBPA caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 350 μg/mL (p<0.01, including gaps), when compared with the vehicle control. No other statistically significant increases were seen. In the presence of S9 mix, HBPA caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 475 and 500 μg/mL (p<0.001, excluding and including gaps), when compared with the vehicle control. No statistically significant increases were seen at 400 μg/mL.

A statistically significant increase in polyploid metaphases was observed during metaphase analysis at 350 μg/mL (p<0.01, in the absence of S9 mix) and 475 and 500 μg/mL (p<0.001, in the presence of S9 mix) when compared to the concurrent vehicle controls. A quantitative analysis for polyploidy using 500 metaphase cells (where possible) was also made for all cultures used in chromosomal analysis. A statistically significant increase (p<0.001) in the proportion of polyploid cells was seen when compared to the vehicle control at 350 (in the absence of S9 mix) and at 475 and 500 μg/mL (in the presence of S9 mix).

 

It is concluded that HBPA 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. HBPA 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.

 

 

HBPA was tested (Huntingdon Life Sciences, 2012, Study MOG0012) for potential mutagenicity in the mouse lymphoma L5178Y cell mutation test. The study was conducted according to EU Method B.17, OECD test guideline 476, EPA test guideline OPPTS 870.5300, and in compliance with GLP.

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

 

Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to HBPA at concentrations from 4.7 to 2404 μg/mL, relative suspension growth (RSG) was reduced from 122 to 0% and from 114 to 0% in the absence and presence of S9 mix, respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 113 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 250 and 350 μg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 24 and 5% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 175 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 14%.

It was concluded that HBPA did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Justification for classification or non-classification

In an in vitro cytogenetic test system it was concluded that Rikabinol HB was positive, under the experimental conditions described.