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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 2009 till 16 March 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to a OECD 487 guideline and in compliance with GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
The result was not confirmed in an independent repeat test
Principles of method if other than guideline:
The test was performed in agreement with OECD 476, using the fluctuation method: after exposure, cytotoxicyty, cloning efficiency and mutation frequency were determined by seeding know numbers of cells into wells of microtiter plates.
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
L-Lysine HCl
L-Lysine HCl
Details on test material:
- Name of test material (as cited in study report): Lysine HCl
- Description: L-lysine-monohydrochloride, technically pure
- Substance type: Powder, white to pale yellow crystals
- Physical state: Solid
- Analytical purity: 97.5%
- Purity test date: 11-04-2008
- Lot/batch No.: April 2008
- Expiration date of the lot/batch: 30 April 2011
- Stability under test conditions: no data
- Storage condition of test material: room temperature, in the dark


Target gene:
Thymidine Kinase locus on chromosome 11.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse-serum (10% for flasks and 20% for microtiter plates).
- 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:
other: doubling time and viability were checked on the day of expousre (12.3 h and 97% respectively)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of male Wistar rats induced with Aroclor 1254
Test concentrations with justification for top dose:
1.2, 2.5, 4.9, 7.0 and 10 mmol/L (with and without S9); 10 mmol/L is equivalent to 1830 µg/mL; concentrations were corrected for test substance purity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: the test material was soluble in culture medium
Details on test system and experimental conditions:
METHOD OF APPLICATION: in culture medium

- Preincubation period: not applicable
- Exposure duration: 24 hours (-S9) and 4 hours (+S9)
- Expression time (cells in growth medium): 44-48 hours
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 tubes per concentration and the solvent control containing 3x10^6 cells (-S9) or 5x10^6 cells (+S9) were treated (one tube for the positive controls)

NUMBER OF CELLS EVALUATED: per replicate tube for selection of mutants 2000 cells were seeded into each well of two 96-well plates in the presence of TFT. After incubation for 10-14 days, the number of wells without cell growth was counted.

DETERMINATION OF CYTOTOXICITY: During expression, cytotoxicity was determined by counting the number of cells after 20-24 hours and 44-48 hours. Survival after expression (cloning efficiency) was determined by adding a 0.2 mL aliquot of a culture diluted to 10 cells/mL into each well of two 96-well plates; after incubation for 10-14 days, the number of wells without cell growth was counted.

- At the start and end of treatment, all cell cultures were checked visually and selected cultures were checked for viability using trypan blue exclusion. In the gene mutation analysis, the number of wells containing large colonies and the number containing small colonies were scored for the positive controls. This was not done for the test substance since no increase in mutant frequency was observed.
Evaluation criteria:
The assay was considered valid if the following criteria were met: (1) The cloning efficiencies at Day 2 in the untreated control cultures in the absence of S9 metabolism fell within the range of 60-140%. (2) The untreated control growth factor over 2 days fell within the range of 8-32. (3) The mutant frequencies in the untreated control cultures fell within the range of 40-300 per 10^6 viable cells. (4) The mutant frequency of the positive control chemicals should be more than 400 per 10^6 viable cells and at least twice that of the corresponding negative control.
A response was considered to be positive if the induced mutant frequency (mutant frequency of test substance minus that of the vehicle negative control) was more than 126 per 10^6 viable cells.
The test item was considered to be mutagenic if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
See below.
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data in report
- Precipitation: not reported
- Other confounding effects: no data




Remarks on result:
other: other: gene mutation at TK locus
Migrated from field 'Test system'.

Any other information on results incl. tables

At the start and end of treatment, all cell cultures were checked visually and no abnormalities were observed. Selected cultures were checked for viability using trypan blue exclusion, and at the highest concentration viability was >90%. A summary of the remaining results is shown in the table below.


Table 1.               Results (mean values) for mouse lymphoma forward mutation test


without S9 without S9 with S9 with S9
dose (mmol/L) mutation frequency % relative total growth mutation frequency % relative total growth
10 85 105 56 93
7 124 94 69 95
4.9 82 110 83 86
2.5 87 96 98 102
1.2 86 104 60 86
0 96 100 50 100
pos. control 497 34 742 37

Mutation frequency: per 10^6 cells

% Relative Total Growth: based on total growth factor during the 2-day expression and cloning efficiency after the 2-day expression.

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative The negative response was not confirmed in an independent repeat test.

In a test according to OECD 476, the test material was not mutagenic at the TK locus of L5178 mouse lymphoma cells, both in the presence and absence of S9.