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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

L-valine did not exhibit genetic toxicity in several in vitro GLP guideline studies:

- OECD 473 (In vitro Mammalian cell Chromosome Aberration Test)

- OECD 476 In vitro Mammalian cell Gene Mutation Test

- OECD 471 Bacterial Reverse mutation Assay (Ames test)

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
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 / 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.
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.
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'.
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

L-valine was tested for genetic toxicity in 3 in vitro GLP guideline studies of different levels. Neither the Ames-test (OECD 471) nor the mammalian cell gene mutation test (OECD 476) nor the mammalian cell chromosome aberration test (OECD 473) did exhibit any genetic toxicity.

Older studies with the Ames-test and a calculation model to investigate sister chromatid exchanges and chromosomal aberrations in chinese hamster ovary cells showed the same negative result for genetic toxicity.

These results were foreseeable as L-valine is a naturally occurring essential amino acid. L-valine 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. L-valine is present in significant amounts in human body fluids – e. g. human blood plasma (Cynober 2002) - as well as in human cells. It is a basic metabolite and building block of all living organisms and therefore a genotoxic/mutagenic potential could be excluded.

 

Cynober L (2002): Plasma Amino Acid Levels With a Note on Membrane Transport: Characteristics, Regulation, and Metabolic Significance. Nutrition 18 (9), 761-766

Justification for selection of genetic toxicity endpoint

Additional key studies referring to several endpoints of REACH Annexes VII - VIII are: V 6203/04_2005 and V 6205/06_2005.

Justification for classification or non-classification

 L-valine is negative in several in vitro mutation tests.
Furthermore, as L-valine is a ubiquitously occuring substance in food,
the environment and even in human body fluids there is no concern
with respect to mutagenicity. L-valine should not be classified as a mutagen
(as a germ cell mutagen in the wording of CLP).