L-valine

Genetic toxicity: in vitro

Administrative data

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

Study period:

2005-03-16 to 2005-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

L-valine

EC Number:

200-773-6

EC Name:

L-valine

Cas Number:

72-18-4

Molecular formula:

C5H11NO2

IUPAC Name:

L-valine

Method

Target gene:

The objective of OECD guideline 473 and of this study was to provide data on the ability of the test substance L-valine to induce structural chromosomal aberrations in cultured Chinese Hamster Ovary (CHO) cells.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 (with Glutamax-I), penicillin-streptomycin (100 IU/ml) and foetal calf serum (10 %). Source: Life Technologies (Gibco) B.V., Breda, The Netherlands
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First chromosomal aberration test: Concentrations in the culture medium of 1172, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 and 2 µ/ml.
Second chromosomal aberration test: Concentrations in the culture medium of 1172, 900, 700, 500, 300, 200, 100, 75, 50 and 25µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium
- Justification for choice of solvent/vehicle: Required by method.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

serum-free culture medium

Positive controls:

yes

Positive control substance:

other: Absence of S9-mix: mitomycib C. Presence of S9-mix: cyclophosphamide

Remarks:

Same control substances for first and second chromosomal aberration test.

Details on test system and experimental conditions:

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 and then incubated at ca. 37 °C in humidified air containing 5 % C02. 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 % C02 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 % C02. 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 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 % C02. Two hours before the end of the culture period (18 hours), the cells and culture medium were checked visually.


Second (independent) chromosomal aberration test
The dose Ievels, 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 the first chromosomal aberration test. 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.

Evaluation criteria:

The study was considered valid because the positive controls gave the statistically significant increases in the number of aberrant cells and the negative controls were within the historical range.

A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cells with structural chromosomal
aberrations is observed.

A response is considered to be equivocal if the percentage of cells with structural chromosomal aberrations is statistically marginal higher than that of the negative control (0.05
A test substance is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberrations over the
concurrent control frequencies is observed, or if a single positive test point is observed in both tests.

A test substance is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points.

Cells with only gaps (achromatic lesions), heavily damaged cells (cells with multiple aberrations) and cells with polyploidy and endoreduplication were
recorded separately and not induded in the final assessment of clastagenic activity.

Both statistical significance and biological relevance are considered together in the evaluation of the results.

Statistics:

Data were analysed statistically by Fisher's exact probability test (two-sided) to determine significant differences between treated and control cultures.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first chromosomal aberration test, in the pulse treatment group with metabolic activation (S9-mix), the mitotic indices of none of the concentrations
analysed (250, 500 and 1172 µg/ml) were reduced, when compared to the mitotic index of the concurrent (culture medium) control. 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

In the first chromosomal aberration test, in the pulse treatment group without metabolic activation (S9-mix), the mitotic indices of the highest (1172 µg/ml) and lowest concentration (250 µg/ml) analysed were reduced to 91 % and 85 %, respectively of that of the concurrent (serum-free culture medium) control. The
mitotic index of the moderate concentration (500 µg/ml) analysed was not reduced, when compared to the rnitotic index of the concurrent (serum-free culture medium). 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.

In the second chromosomal aberration test, in the pulse treatment group with metabolic activation (S9-mix), the mitotic indices of the concentrations analysed
(700, 900 and 1172 µg/ml) were reduced to 85 %, 88 % and 78 %, respectively of that of the concurrent (serum-free culture medium) control. 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.


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 (700, 900 and 1172 µg/ml) were reduced to 88 %, 84 % and 92 %, respectively of that of the concurrent (serum-free culture medium)
control. 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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