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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2017 - 21 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Physical State / Appearance: Red Powder
Batch: 021340
Storage Conditions: Room temperature, in the dark

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Harlan CCR
- Suitability of cells: The high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The maximum recommended dose level was 5000 μg/mL however the maximum achievable dose level was 2500 μg/mL due to formulation issues at the MRD.
0.02 to 2 μg/mL in the absence of metabolic activation and 0.03 to 3 μg/mL in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1 x 10e7 cells/225 cm2 flask approximately 24 hours before dosing

DURATION
- Preincubation period: 24 hoiurs
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Rationale for test conditions:
prliminary experiment results
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
Statistics:
Student’s t-test

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at 2 μg/mL in the absence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.
- Precipitation: No precipitate of the test item was observed throughout.

RANGE-FINDING/SCREENING STUDIES: No precipitate of the test item was observed in either of the exposure groups. The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines.

Applicant's summary and conclusion

Conclusions:
Lumière Pink S.M. 8135N did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.