Lysine hydrochloride

Genetic toxicity: in vitro

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:

17 February 2009 till 16 March 2009

Reliability:

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

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

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

Test material information

Constituent 1

Reference substance name:

Lysine hydrochloride

EC Number:

211-519-9

EC Name:

Lysine hydrochloride

Cas Number:

657-27-2

Molecular formula:

C6H14N2O2.ClH

IUPAC Name:

lysine hydrochloride

Method

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

DURATION
- 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.

OTHER EXAMINATIONS:
- 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.

Statistics:

None.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See below.
TEST-SPECIFIC CONFOUNDING FACTORS
- 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

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: not relevant

ADDITIONAL INFORMATION ON CYTOTOXICITY: see table below.

Remarks on result:

other: other: gene mutation at TK locus

Remarks:

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

Conclusions:

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.