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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
28 February to 28 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken at a GLP accredited laboratory, to internationally accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
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:
other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH (1996) Guideline S2A and S2B: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
LumiNova Green
IUPAC Name:
LumiNova Green
Constituent 2
Reference substance name:
Dialuminium x dysprosium y europium (1-x-y) strontium tetraoxide
IUPAC Name:
Dialuminium x dysprosium y europium (1-x-y) strontium tetraoxide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): LumiNova Green
- Substance type: Inorganic phosphorescent pigment
- Physical state: Powder
- Analytical purity: > 99.0%
- Impurities (identity and concentrations): No data
- Purity test date: 24 January 2011
- Lot/batch No.: PM-305
- Expiration date of the lot/batch: 21 January 2012
- Stability under test conditions: No data
- Storage condition of test material: Room temperature (ca. 20ºC), in the dark
- Other:

Method

Target gene:
Thymidine kinase locus (TK+/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.94, 1.88, 3.75, 7.5, 15 and 30 μg/mL
Vehicle / solvent:
- Vehicle; water
- Justification for choice of solvent/vehicle:
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
In the absence of S9 mix
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); in suspension;

DURATION
- Preincubation period:
- Exposure duration: 3h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

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

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: E05 to E06

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

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

OTHER:
Evaluation criteria:
Tests were accepted on the basis of the following criteria:

Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 μg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3 hour treatments and between 32 to 180 on Day 2 following a 24 hour treatment.
Obvious outliers were excluded. However, there were at least 2 vehicle controlcultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
There was not excessive heterogeneity between replicate cultures.

Assays that did not fulfil the required criteria were rejected and therefore are not reported. This decision was at the discretion of the Study Director.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test substance (formerly reported as LumiNova) was previously determined to be soluble at 0.3 g/L (0.3 mg/mL) in water at ca. 20ºC (Huntingdon Research Centre Ltd., Study Number NMO 6/951713), with acceptable pH values for this test system. Test solutions had been shown to become increasingly alkaline at higher concentrations. Based on the limit solubility of LumiNova Green, the previously generated data was used to achieve a final concentration of 30 μg/mL, dosed at 10% v/v. This was the maximum concentration in the
preliminary toxicity test. Justification for the highest selected concentration was based on achieving the maximum solubility within acceptable pH parameters in this test system.
- Effects of osmolality: The osmolality and pH of the test substance in medium was tested at the highest tested final concentration of 30 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg, and no fluctuations in pH of the medium of more than 1.0 unit were observed, when compared with the vehicle control.
- Evaporation from medium: No data
- Water solubility: 300 mg/l
- Precipitation: No
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: No precipitate was observed by eye at the end of treatment. Exposure to LumiNova Green at concentrations from 0.06 to 30 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in no reduction in RSG at any LumiNova Green test concentration. Following a continuous exposure for 24 hours, no precipitate was observed by eye at the end of treatment at any concentration tested. Exposure to concentrations from 0.06 to 30 μg/mL resulted in no reduction in RSG at any LumiNova Green test concentration.
Concentrations used in the main test were based upon these data.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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