Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
L-isoleucine
EC Number:
200-798-2
EC Name:
L-isoleucine
Cas Number:
73-32-5
Molecular formula:
C6H13NO2
IUPAC Name:
L-isoleucine
Test material form:
solid: crystalline

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The mouse lymphoma L5178Y cells (L5178Y tk +/- 3.7.2C line), used in the gene mutation assay, were obtained from Dr. J. Cole, MRC Cell Mutation Unit, University of Sussex, United Kingdom. The chromosome number of these cells is 40 (stable aneuploid karyotype, 2n = 40). The cells were stored as frozen stock cultures in liquid nitrogen. Subcultures were prepared from these stocks (stock from 12 July 2002) for experimental use. Each new stock culture is checked for mycoplasma contamination, which was absent.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
9.5, 6.6, 4.6, 2.3, 1.2, and 0.58 mmol/L.
Vehicle / solvent:
The test substance was dissolved in serum-free culture medium.
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
Cell treatment without metabolic activation
In the assay without metabolic activation the cells were exposed to the test substance according to the following procedure! 4 mL Test substance solution or negative control and 1 mL culture medium (without horse serum), or 100 µL positive control and 4.9 ml culture medium (without horse serum) were added to ca. 3,000,000 L5178Y cells in 5 mL culture medium (with 10 % horse serum) to a final volume of 10 mL. Two cultures treated with the vehicle (culture medium without serum) were used as negative controls; one single culture treated with MMS was used as positive control substance at a final concentration of 0.1 mmol/L. Duplicate cultures were used for each concentration of the test substance. The cells were exposed for 24 h at ca. 37 °C and ca. 5 % CO2 in a humidified incubator.
The dose levels of the test substance used ranged from 9.5 to 0.58 mmol/L L-Isoleucine. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

Cell treatment with metabolic activation
In the assay with metabolic activation the cells were exposed to the test substance according to the following procedure. 4 mL Test substance solution or negative control, or 100 µL positive control and 3.9 mL culture medium (without serum), and 1 mL 20 % (v/v) S9-mix were added to ca. 5,000,000 L5178Y cells in 5 ml culture medium (with 10 % horse serum) to a final volume of 10 mL. Two cultures treated with the vehicle (culture medium without serum) were used as negative controls; one single culture treated with MCA was used as positive control substance at a final concentration of 10 µg/mL. Duplicate cultures were used for each concentration of the test substance. The cells were exposed for 4 h at ca. 37 °C and ca. 5 % CO2 in a humidified incubator.
The dose levels of the test substance ranged from 9.5 to 0.58 mmol/L L-isoleucine. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

Assessment of cytotoxicity
The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG). The relative initial cell yield is the ratio of the amount of cells after treatment to that of the vehicle control and is a measure for growth during treatment. The RSG is a measure for the cumulative growth rate of the cells 24 h and 48 h after treatment compared with untreated control cultures; the RTG is the product of the relative initial cell yield, the RSG and the relative colony-forming ability ('cloning efficiency') of the cells 48 h after treatment compared with negative control cultures, and is a measure for cytotoxicity that occurs in all phases of the assay.
After the treatment period, the cultures were checked for visibly aberrant effects (eg. flocculation/precipitation of the test substance and lysed cells), and the viability of the cells treated with the higher concentrations of test substance was checked. The medium containing the test substance, negative control or positive control was removed and the cells were washed twice with culture medium (with 10% horse serum). Finally, the cells were resuspended in culture medium (with 20% horse serum) and the number of cells was counted. The cell suspensions were diluted to 200,000 cells per ml and the cultures were incubated for about 44-48 h at ca. 37°C and ca. 5% CO2 in a humidified incubator to allow near-optimal phenotypic expression of induced mutations.
After 20-24 h and 44-48 h the number of cells of all remaining cultures was counted. After 20-24 h the cell suspensions were diluted, if required, to 200,000 cells per ml and further incubated at ca. 37°C and ca. 5% CO2 as described above. After 48 h a portion of the cells was diluted to 10 cells per ml for determining the cloning efficiency. The remaining cells were used for determining the frequency of TFT-resistant mutants. Portions (200 µL) of each dilution at 10 cells per ml were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5% CO2 in a humidified incubator.
After this period the number of wells without growth of cells was counted and the cloning efficiency was determined using the zero term of the Poisson distribution (Cole et ai, 1983). The ratio of the cloning efficiency of cells treated with the test substance or the positive control compared to that of the vehicle control yields the relative cloning efficiency (RCE). The suspension growth (SG) was calculated. The ratio of the SG of treated cells to that of the vehicle control yields the relative suspension growth (RSG). The relative total growth (RTG) is adjusted for growth during treatment to obtain a measure for cytotoxicity that occurs in all phases of the assay. Reduction of the cell count after treatment, or of the RSG and of the RTG is a measure for the cytotoxicity of the test substance.

Assessment of mutagenicity
One gene mutation assay was performed to evaluate the mutagenic potential of the test substance. This assay was carried out with five concentrations of the test substance in duplicate, two negative controls and one positive control, in both the absence and the presence of the S9-mix.
The frequency of TFT-resistant mutants and the final cloning efficiency of the cells were determined 2 days after starting the test. The number of cells were counted and the final cloning efficiency of the cells were determined. To determine the frequency of TFT-resistant mutants, the cell suspensions were diluted to a density of 10,000 cells per ml in culture medium (with 20 % horse serum) containing 4 µg TFT per mL. Portions (200 µL) of each dilution were transferred to each well of two 96-wells microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5 % CO2 in a humidified incubator.
After this period the number of wells without growth of cells was counted and the cloning efficiency in the TFT plates were calculated. The TK mutant frequency per 1,000,000 clonable cells was finally calculated. The mutant colonies of the negative and positive controls and of some test substance dose levels were scored using the criteria of small and large colonies.
The following definitions were used for colony sizing:
Large colony: covers >25% of the well area; the edge consists of one cell layer
Small colony: covers <25% of the well area; the edge consists of more than one cell layer; the diameter was 1/10 or more of the well



Statistics:
No statistical analysis as performed.

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
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under conditions of the test, L-isoleucine is found to be not mutagenic.
Executive summary:

The test substance L-isoleucine was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and presence of a metabolic activation system (S9 -mix).

One assay was conducted; in this assay five concentrations were tested in duplicate in both the absence and presence of S9-mix. The test substance was dissolved in culture medium without serum prior to testing. The highest concentration tested and evaluated for mutagenicity was 9.5 mmol/L in both the absence and presence of S9 -mix.

In both the absence and presence of S9 -mix L-isoleucine was not cytotoxic to the L5178Y cells. The relative total growth (RTG) at the highest concentration tested was not decreased compared to the negative control.

In both the absence and presence of S9-mix no increase of the mean mutant frequency was observed at any dose level. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; culture medium without serum served as negative control. The negative controls were within acceptable ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, the test substance L-isoleucine is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.