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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:
18 January - 23 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline 476 with minor deviation: In Experiment 1 in the presence of S-9, only three concentrations were analysed for mutant frequency instead of four concentrations.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
In Experiment 1 in the presence of S-9, only three concentrations were analysed for mutant frequency instead of four concentrations.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: MEXORYL SCK
- Physical state: Beige powder
- Analytical purity: saponines content (determined by HPLC assay) 55.8 % w/w
- Lot/batch No.: R0069579A 003 P 001
- Shelf life/Retest date: April 2011
- Storage condition of test material: Stored at room temperature and away from light and moisture

Method

Target gene:
hprt gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Growth media, RPMI 1640
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for spontaneous mutant frequency: Yes
- Other details: For each experiment, one vial was thawed and the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. The cells were allowed to grow well and subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range-finder experiment:
- With and without S9 mix: 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL

Main study:
- Experiment 1: 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 μg/mL, with and without S9 mix
- Experiment 2: 500, 1000, 2000, 2500, 3000, 3500, 3750, 4000, 4500 and 5000 μg/mL, with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water
- Test article solutions were prepared by formulating MEXORYL SCK under subdued light conditions in purified water (with the aid of vortex mixing, warming at 37 ºC, ultrasonication and stirring as required) immediately prior to assay to give the maximum required treatment solution concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 filter, 0.2 μm pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within 1.5 h of initial formulation of the test article.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
treatments with the vehicle purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.1 and 0.15 μg/mL; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
treatments with the vehicle purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
2 and 3 μg/mL; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In RPMI 1640 media (RPMI A, RPMI 5, RPMI 10, RPMI 20)

DURATION
- Exposure duration: 3 h, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Viability scoring: 8 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12-14 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG) at 15 µg/mL (final concentration)

NUMBER OF REPLICATIONS:
- Vehicle control and test substance groups: Single culture for cytotoxicity study and duplicate cultures for experiment 1 and 2.
- Positive control: Single culture for experiments 1 and 2.

NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6-TG resistance respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival (%RS)

OTHER:
- Wells containing viable clones were identified by eye using background illumination and counted.
- Cell concentrations in the selected cultures were determined using a Coulter counter.
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hypoxanthine phosphoribosyl transferase (hprt) locus in mouse lymphoma L5178Y cells if:
- the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
- there was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
- the effects described above were reproducible.

- Results that only partially satisfy the assessment criteria described above were to be considered on a case-by-case basis.
Statistics:
- Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: Test item was soluble in purified water at concentrations up to at least 54.30 mg/mL.
- Effects of osmolality and pH: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (5000 μg/mL) as compared to the concurrent vehicle controls.
Precipitation:
- Range-finder experiment: Following the 3 h treatment incubation period, precipitate was observed at 5000 μg/mL in the absence of S-9 only.
- Experiment 1: Following the 3 h treatment incubation period, precipitate was observed at the highest three concentrations tested in the absence of S-9 (4000 to 5000 μg/mL) and at the highest two concentrations tested in the presence of S-9 (4500 and 5000 μg/mL).

RANGE-FINDING/SCREENING STUDIES:
- Six concentrations were tested, in the absence and presence of S9, ranging from 156.3 to 5000 μg/mL.
- Highest concentration analysed was 5000 μg/mL, which gave 15 % and 76 % RS in the absence and presence of S-9, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with the historical data of the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiment 1 ten concentrations, ranging from 500 to 5000 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment the highest concentrations analysed for viability and 6TG resistance were limited by post-treatment precipitate to 4000 μg/mL in the absence of S-9 and 4500 μg/mL in the presence of S-9, which gave 30 % and 83 % RS, respectively.
- In Experiment 2 ten concentrations, ranging from 500 to 5000 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment the highest concentration analysed for viability and 6TG resistance was 5000 μg/mL in the absence and presence of S-9, which gave 18 % and 86 % RS, respectively.
Remarks on result:
other: other: L5178Y tk +/- (3.7.2C) mouse lymphoma cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

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

Under the test conditions, MEXORYL SCK is not considered as mutagenic at the hprt locus of L5178Y mouse lymphoma cells according to the Annex VI of the Directive 67/548/EEC and the Regulation (EC) N° 1272-2008 (CLP).
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, mouse lymphoma L5178Y tk+/- cells were exposed to MEXORYL SCK dissolved in purified water in RPMI 1640 medium for 3 h, with and without metabolic activation (2 % S9 fraction of male Sprague Dawley rats liver induced with Aroclor 1254), at the following concentrations:

 

Range-finder experiment:

- With and without S9 mix: 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL

 

Main study:

- Experiment 1: 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 μg/mL, with and without S9 mix

- Experiment 2: 500, 1000, 2000, 2500, 3000, 3500, 3750, 4000, 4500 and 5000 μg/mL, with and without S9 mix

 

In the cytotoxicity study, the highest concentration analysed was 5000 μg/mL, which gave 15 % and 76 % RS in the absence and presence of S-9, respectively. In experiment 1, the highest concentrations analysed for viability and 6TG resistance were limited by post-treatment precipitate to 4000 μg/mL in the absence of S-9 and 4500 μg/mL in the presence of S-9, which gave 30 % and 83 % RS, respectively. In experiment 2, the highest concentration analysed for viability and 6TG resistance was 5000 μg/mL in the absence and presence of S-9, which gave 18 % and 86 % RS, respectively.

 

In Experiment 1 in the absence and presence of S-9 and Experiment 2 in the presence of S-9 no statistically significant increases in mutant frequency were observed following treatment with R0069579A at any concentration tested and there were no significant linear trends. In Experiment 2 in the absence of S-9, a statistically significant increase in mutant frequency was observed at a single concentration (4500 μg/mL) and there was a weak (but statistically significant) linear trend (p < 0.05). However, the mutant frequency value at this concentration was 4.86 mutants/106 viable cells, which was within the range of historical control data. This increase was therefore considered sufficiently small in magnitude to be of no biological relevance.Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [4-nitroquinoline 1-oxide at 0.10 or 0.15 µg/mL (without S9 mix) and benzo(a)pyrene at 2 or 3 µg/mL (with S9 mix)] indicating the validity of the study.

 

Under the test conditions, MEXORYL SCK is not considered as mutagenic at the hprt locus of L5178Y mouse lymphoma cells according to the Annex VI of the Directive 67/548/EEC and the Regulation (EC) N° 1272-2008 (CLP).