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Genetic toxicity in vitro

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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:
18 August to 6 September 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in a GLP compliant laboratory according to accepted regulatory guidelines
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/ß-Naphthoflavone induced, rat-liver S9.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Acetone
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 5000 µg/plate
METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period:N/a
- Exposure duration: 48h
- Expression time (cells in growth medium):N/a
- Selection time (if incubation with a selection agent): N/a
- Fixation time (start of exposure up to fixation or harvest of cells): N/a

NUMBER OF REPLICATIONS: 3 plates at each cocentration against each tester strain


DETERMINATION OF CYTOTOXICITY
- Method: the frequency of revertant colonies assessed using a Domino colony counter.

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges
are presented in the standard test method of this report with historical control ranges up to and including 1997 and 1998 presented in an Appendix to this report.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures were in the range of 1 to 9.9 x 10-9 bacteria per ml.
Each mean positive control value was at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges up to and including 1997 and 1998 are presented in an Appendix to this report.
Statistics:
(Dunnett's method of linear regression(5)
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:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations:
Solvent control plates gave counts of revertant colonies
within the normal range.

All positive control chemicals gave increases in revertants,
either with or without the metabolising system as
appropriate, within expected ranges. No statistically
significant increase in the number of revertant colonies was
recorded for any of the bacterial strains with any dose of
the substance, either with or without metabolic activation.
The substance was found to be non-mutagenic under the
conditions of the test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-mutagenic under the conditions of this
test.
Executive summary:

The test material was considered to be non-mutagenic under the conditions of this test.

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:
22nd October 1999 to 14th January 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: STUDY CONDUCTED IN A GLP COMPLIANT LAB ACCORDING TO APPROVED REGULATORY GUIDELINES
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Annex V (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Species / strain / cell type:
other: Chinese Hamster Lung (CHL) cells.
Details on mammalian cell type (if applicable):
- Type and identity of media:The Chinese Hamster Lung (CHl) cell line, isolated by Koyama et al (1970) and cloned by Ishidate and Sofuni (1985), was used.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically "cleansed" against high spontaneous background: not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone and ß-Naphthoflavone induced rat liver S9.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 78.13 µg/ml
Concentration range in the main test (without metabolic activation): 312.5 µg/ml
Vehicle / solvent:
Acetone
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not specified
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 6 hours
Exposure period (without metabolic activation): 6 hours

Fixation time:
18 hours.
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidel ines for mutagenicity testing (Appendix IV).
Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells are recorded and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately. The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported.
The number of gap-type aberrations was recorded.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 625 µg/ml)
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(78.13 µg/ml)
Additional information on results:
Observations:
A precipitate of the test material was observed at the end
of the treatment period at 312.5µg/ml in the absence of S9
but was not observed at any dose level in the presence of
S9.


No significant or dose-related increases in the frequency of
cells with aberrations were seen in any of the treatment
cases.


All positive control treatments induced a significant
increase in the frequency of abberations. Negative control
values were within the normal range.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material, S-900, did not induce any statistically significant, dose-related increases in the frequency of cells with chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times. S-900 is therefore considered to be non-clastogenic to CHI. cells in vitro.
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:
21st May to 17th June2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted in GLP compliant laboratiry according to accepted regulatory Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
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
Deviations:
no
Qualifier:
according to
Guideline:
other: US FDA Center for Food Safety & Applied Nutrition (2006) Redbook 2000: Toxicological Principles for the Safety Assessment of Food Ingredients.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: donor horse serum (HiDHS) containing 10% DMSO
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 9.766, 19.531, 39.063, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 ug/mL
Mutation tests: -
S9 mix (3 hours) 0.01, 0.1, 1, 5, 10, 20, 30, 40 and 50 ug/mL
+S9 mix (3 hours) 10, 20, 50, 75, 150, 300, 450 and 600 ug/mL
-S9 mix (24 hours) 0.001, 0.01, 0.1, 1, 5, 10, 20 and 30 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone;
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in vehicles compatible with this test system were assessed. S-900 was found to be soluble at 500 mg/mL in acetone. A solution of 500 mg/mL, dosed at 1% in medium, showed no precipitate in the culture medium.
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
DURATION
- Preincubation period: N/A
- Exposure duration: 3h in the absence and presence of S9 mix, 24 hours in the absence of S9 mix
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 11 days for 3 hour exposure, 10 days for 24 hour exposure
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): trifluorothymidine
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls.

NUMBER OF CELLS EVALUATED: 2 x 103 cells/well, 96 wells per plate, 2 plates per culture, 2 cultures per concentration analysed (4 cultures for vehicle control).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Relative Total Growth

OTHER EXAMINATIONS:
Evaluation criteria:
The number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF).
The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants.
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.
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 specified
Positive controls validity:
valid
Conclusions:
It was concluded that S-900 did not demonstrate mutagenic potential in this in vitro cell
mutation assay, under the experimental conditions described.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

It was concluded that S-900 did not demonstrate mutagenic potential in the three in vitro cell mutation assays, under the experimental conditions described