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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
For each of the genotox endpoints at least one in-vitro test according to GLP and OECD guidelines is available: an Ames test, an in-vitro chromosome aberration test and a gene mutation study in L5178 cells. L-isoleucine was found to be negative in each of these tests. As a consequence, it can be concluded that L-isoleucine is not genotoxic.
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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
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
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.
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.
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
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Physical state: white powder
- Analytical purity: 94.2% (w/w)
- Lot/batch No.: 1
- Expiration date of the lot/batch: 1 December 2007
- Storage condition of test material: ambient temperature, dark
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO cells (CHO K-l line) were obtained from Prof. Dr. AT. Natarajan, University of Leiden, The Netherlands. This cell line derives from the CHO cells originally isolated from an explant of the ovary of the Chinese hamster (Cricetulus griseus, 2n = 22) by Kao and Puck (1968). The modal chromosome number of these cells is 20-22 (stable aneuploid karyotype). The cell-cycle time is 12-14 h. The cells are stored as frozen stock cultures in liquid nitrogen. Subcultures were prepared from these stocks (passage 16) for experimental use. Each passage CHO cells in the liquid nitrogen is checked for mycoplasma contamination and karyotype stability, which were absent and stable, respectively.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Final concentrations in the culture medium in the first chromosomal aberration test:
1312, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 and 2 µg/mL.

Final concentrations in the culture medium in the second chromosomal aberration test:
1312, 1000, 800, 600, 400, 300, 200, 100, 50 and 25 µg/mL.
Vehicle / solvent:
The test substance was dissolved in serum-free culture medium. This resulted in a clear solution which was sterilized by passage through a 0.45 urn filter.
Untreated negative controls:
yes
Remarks:
serum-free culture medium
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
The first chromosomal aberration test
Exponentially growing cells were seeded in sterile, screw-capped tissue culture flasks (surface area 25 cm2; 120,000 cells per flask) containing 5 ml culture medium (see paragraph 2.4) and then incubated at ca. 37 °C in humidified air containing 5 % CO2. On the next day (ca. 24 hours after seeding), the cells were exposed to the test substance, in both the absence and presence of the S9-mix. In all instances duplicate cultures were used.
In the absence of the S9-mix, 0.5 ml of the vehicle control (serum-free culture medium), 0.5 ml of each of the dilutions of the test substance or 50 µl of the positive control substance mitomycin C was added to the tissue culture medium in the flasks and the culture medium was checked visually. The total volume in the flasks was 5 ml. The cultures were incubated at ca. 37 °C in humidified air containing ca. 5 % CO2 and treated for 4 hours (pulse treatment). After 4 hours, the cells and culture medium were checked again. The medium with the test substance was removed, the cells were washed twice with phosphate-buffered saline (pH 7.4) and supplied with 5 ml freshly prepared culture medium. Thereafter, the cells were incubated for an additional 14 hours at ca. 37 °C in humidified air containing ca. 5 % CO2. Two hours before the end of the culture period (18 hours), the cells and culture medium were checked visually.
In the presence of the S9-mix, the culture medium with foetal calf serum was replaced by culture medium with penicilline and streptomycine but without foetal calf serum. Thereafter, 0.5 ml of the vehicle control (serum-free culture medium), 0.5 ml of each of the dilutions of the test substance or 50 µl of the positive control substance cyclophosphamide was added to the tissue culture medium in the flasks and the culture medium was checked visually. Thereafter, 0.5 ml of S9-mix (see paragraph 2.5) was added to all cultures. The total volume in the flasks was 5 ml. After 4 hours, the culture medium and the cells were checked visually. The medium with the test substance was removed, the cells were washed twice with phosphate-buffered saline (pH 7.4) and supplied with 5 ml freshly prepared culture medium with foetal calf serum. The cells were incubated for an additional 14 hours at ca. 37 °C in humidified air containing ca. 5 % CO2. Two hours before the end of the culture period (18 hours), the cells and culture medium were checked visually.

The second (independent) chromosomal aberration test
The dose levels, used in the second chromosomal aberration test, were based on the results obtained in the first chromosomal aberration test. The second chromosomal aberration test was carried out essentially as described above. In the presence of S9-mix, the cells were pulse-treated for 4 hours. In the absence of S9-mix, the cells were treated continuously for 18 hours. The cells of both treatment groups were harvested 18 hours after onset of the treatment.
Statistics:
Data were analysed statistically by Fisher's exact probability test (two-sided) to determine significant differences between treated and control cultures.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

In the first chromosomal aberration test, in both pulse treatment groups with and without metabolic activation (S9-mix), the mitotic indices of none of the concentrations analysed (250, 500 and 1312 /xg/ml) were reduced, when compared to the mitotic index of the concurrent (serum-free culture medium) control cultures. In both treatment groups, the test substance did not induce a statistically significant increase in the number of aberrant cells, when compared to the number of aberrant cells found in the negative (serum-free culture medium) control cultures.

In the second chromosomal aberration test, in the pulse treatment group with metabolic activation (S9-mix), the mitotic indices of none of the concentrations analysed (800, 1000 and 1312 fig/ml) were reduced, when compared to the mitotic index of the concurrent (serum-free culture medium) control culture. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells, when compared to the number of aberrant cells found in the negative (serum-free culture medium) control culture.

In the second chromosomal aberration test, in the continuous treatment group of 18 hours without metabolic activation (S9-mix), the mitotic indices of the concentrations analysed (800, 1000 and 1312 jig/ml) were reduced to 89 %, 87 % and 87 %, respectively of that of the concurrent (serum-free culture medium) control culture. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells, when compared to the number of aberrant cells found in the negative (serum-free culture medium) control cultures (see Table 4 and 8). In both chromosomal aberration tests, the positive control substances mitomycin C (in the absence of a metabolic activation system) and cyclophosphamide (in the presence of a metabolic activation system), induced the expected statistically significant increases in the incidence of structural chromosomal aberrations.

Conclusion:

The data obtained in both chromosomal aberration tests, support the conclusion that, under the conditions used in this study, the test substance L-Isoleucine was not clastogenic for CHO cells.

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

Under the conditions of the test, L-Isoleucine was not clastogenic for CHO cells.
Executive summary:

The test substance L-Isoleucine was examined for its potential to induce structural chromosomal aberrations in Chinese Hamster Ovary (CHO) cells, in both the absence and presence of a metabolic activation system (S9-mix). Two chromosomal aberration tests were conducted. The maximum final concentration in the culture medium (1312 µg/mL; 10 mmol/L), used in both chromosomal aberration tests, was based on the molecular weight (131.17) of the test substance, as stipulated in the study plan.

In the first chromosomal aberration test, in both the absence and presence of a metabolic activation system (S9-mix), the treatment/harvesting time was 4/18 hours (pulse treatment). In both the absence and presence of S9-mix, three concentrations of the test substance (1312, 500 and 250 µg/mL) were analysed for chromosomal aberrations.

In the second chromosomal aberration test, in the presence of S9-mix, the treatment/harvesting time was 4/18 hours (pulse treatment). In the absence of S9-mix, the treatment/harvesting time was 18/18 hours (continuous treatment). In both the presence and absence of S9-mix, three concentrations of the test substance (1312, 1000 and 800 µg/mL) were analysed for chromosomal aberrations.

In both the first and second chromosomal aberration test, the test substance L-Isoleucine did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells found in the negative control (culture medium) cultures.

In both chromosomal aberration tests, the numbers of aberrant cells, found in the negative control (culture medium) cultures, were within the historical range and the positive control substances mitomycin C (in the absence of the S9-mix) and cyclophosphamide (in the presence of the S9-mix) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations. This demonstrates the validity of both chromosomal aberration tests.

These data support the conclusion that, under the conditions used in this study, the test substance L-Isoleucine was not clastogenic for Chinese Hamster Ovary (CHO) cells.

Endpoint:
in vitro gene mutation study in bacteria
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): L-isoleucine
- Physical state: white powder
- Analytical purity: 94.2%
- Lot/batch No.: 1
- Storage condition of test material: ambient temperature, dark
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 62, 185, 556, 1667, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: saline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: N-ethyl-N-nitrosourea
Details on test system and experimental conditions:
To 2 mL molten top agar, maintained at 46°C, were added subsequently: 0.1 mL of a fully grown culture of the appropriate strain, 0.5 mL of the test substance, or of the negative or 0.1 mL of the positive control substance solution, and 0.5 mL of S9-mix for the experiments with metabolic activation or 0.5 mL sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5% agar in Vogel and Bonner medium E with 2% glucose). All determinations were made in triplicate. The plates were incubated at ca. 37°C for approx. 72 hours. Subsequently, His+ and Trp+ revertants were counted. Cytotoxicity is defined as a reduction (at least 50%) in the number of revertant colonies and/or a cleaning of the background lawn of bacterial growth.
Evaluation criteria:
The mutagenicity is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plate is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Both numerical significance and biological relevance are considered together in the evalution.
Statistics:
No statistics are performed.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1667 µg/plate in the absence of S9-mix, at 5000 µg/plate in the absence of S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate in the absence of S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA100, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative

L-isoleucine was not mutagenic under the conditions of the test.
Executive summary:

L-isoleucine was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9 mix).

 

L-isoleucine was dissolved in saline. One bacterial reverse mutation test was performed with all strains in the absence and the presence of S9-mix with 5 different concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (saline) and positive controls were run simultaneously with the test substance.

 

L-isoleucine was slightly toxic to strain TA1537, in the absence of S9-mix at and above 1667 µg/plate and to TA1537 and TA98, in the presence of S9-mix at 5000 µg/plate, as was evidenced by a slightly less dense background lawn of the bacterial growth compared to the negative control.

 

In both the absence and the presence of S9-mix in all strains, L-isoleucine did not cause a dose related or more than two-fold increase in the number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.

 

The mean number of His+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

 

It is concluded that L-isoleucine was not mutagenic under the conditions employed in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A number of genotox tests are available for L-isoleucine: 3 Ames tests, 3 chromosome aberration assays and 2 gene mutation assays.

In the key Ames study (van den Wijngaard, 2005) concentrations up to 5000 µg/plate were tested in the presence and the absence of S9 -mix on S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) strains. L-isoleucine did not cause a dose related response or more than 2 -fold increase in the number of revertant colonies on the test plates comparing to the background of spontaneous reversion rates, observed with the negative control. Therfore, L-isoleucine was found not te be mutagenic under the conditions of the test. The supporting studies (Motoi Ishidate, 1999 and BOZO, 2006) confirmed that L-isoleucine had no bacterial reverse mutagenic activity (negative) under the conditions of these studies.

Regarding chromosome aberration, in the key study ( de Vogel, 2005) L-isoleucine concentrations up to 1312 µg/mL (= 10 mmol/L) were analysed for chromosomal aberrations on Chinese Hamster Ovary (CHO) cells. L-Isoleucine did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells found in the negative control (culture medium) cultures. These data support the conclusion that, under the conditions used in this study, the test substance L-Isoleucine was not clastogenic for Chinese Hamster Ovary (CHO) cells. The two supporting studies (Sofuni, 1999 and Zhang 1992) confirmed this result.

Furthermore, L-isoleucine was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, both in the absence and presence of a metabolic activation system (S9 -mix) (key - Steenwinkel, 2005). The highest concentration tested and evaluated for mutagenicity was 9.5 mmol/L. 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. 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. The supporting study by Sargentini and Smith (1986) supports this conclusion.

In conclusion, based on the above information, L-isoleucine was concluded to not be genotoxic.

These results were foreseeable as L-isoleucine is a naturally occurring essential amino acid. L-isoleucine is a normal constituent in living cells occurring as a free amino acid, bound to RNA and incorporated in proteins and peptides. It is ingested daily in significant amounts. Therefore human exposure through food is orders of magnitude higher than the anticipated levels of exposure from the uses covered by this dossier.

Justification for classification or non-classification

As all in-vitro tests are negative, classification of L-isoleucine for this endpoint is not required.