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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read across from L-arginine to L-arginine-HCl is performed.

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

- OECD 473 (In vitro Mammalian Cell Chromosome Aberration) (2 studies)

- OECD 471 (Bacterial Reverse Mutation Assay; Ames test) (2 studies).

In summary, L-arginine-HCl is not considered to be genotoxic.

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2005-07-27 to 2006-02-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study but read across
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0 mg/l, 6.8 µg/l, 13.59 µg/l, 27.19 µg/l, 54.38µg/l, 108.75 µg/l, 217.5 µg/l, 435 µg/l, 870 µg/l, 1740 µg/l.
Experiments 1 and 3: 435 µg/l, 870 µg/l, 1740 µg/l.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tissue culture medium = Eagle's minimal essential medium with HEPES buffer (MEM).
- Justification for choice of solvent/vehicle: Sustains activity of testing cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION AND OTHER CULTURE CONDITIONS
1. With metabolic activation (S9) treatment:
After 48 h the cultures were transferred to tubes and centrifugated. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 ml of the appropriate solution of vehicle control or test material was added to each culture. For the positive
control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9-mix (ie 2% final concentration of S9 in standard co-factors) was added to the cultures of the preliminary toxicity Test and of experiment 1.
In Experiment 2, 1 ml of 10% S9-mix (ie 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.
After 4 hours, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37°C.
2. Without metabolic activation (S9) treatment:
In Experiment 1, after approximately 48 hours incubation the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium
was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 ml of the appropriate vehicle control, test material solution or 0.1 ml of positive control solution. The total volume for each culture was a nominal 10 ml.
After 4 hours, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture . medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2 the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.0 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 ml of vehicle control, test material dose solution or 0.1 ml positive control solution. The nominal final volume of each culture was 1 0 ml. The cultures were then incubated for 24 hours.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (clocemid 0.1 µg/l).
STAIN (for cytogenetic assays): 5 % Gurrs Giemsa for 5 minutes.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The following numbers apply for experiments 1 and 2 and are per replicate.
Vehicle control, test material, with and without metabolic activation, positive control with metabolic activation: 100
Positive control without metabolic activation: 50 (slide evaluation terminated at 50 cells because approximately 50 % cells with aberrations had been observed.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: human
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:
RANGE-FINDING/SCREENING STUDIES: Yes: preliminary toxicity test

COMPARISON WITH HISTORICAL CONTROL DATA: Yes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In a GLP guideline study according to OECD 473 L-arginine did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The maximum test concentration was 1740 µg/l. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
The results for L-arginine are transferred to L-arginine-HCl by read across.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Read across from L-arginine to L-arginine-HCl is justified for genetic toxicity, too. L-arginine-HCl is present as L-arginine under physiological conditions and in human body fluids.

L-arginine was tested for genetic toxicity in a total of 4 in vitro GLP guideline studies, 2 of them were the bacterial reverse mutation assay, 2 of them were mammalian cell chromosome aberration tests. Neither the Ames-test (OECD 471) nor the mammalian cell chromosome aberration test (OECD 473) did exhibit any genetic toxicity.

These results were foreseeable as L-arginine is a naturally occurring amino acid. L-arginine 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-arginine 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

An additional key study referring to the endpoint 8.4.1 of REACH Annex VII is: 2148/001.

Justification for classification or non-classification

L-arginine is negative in several in vitro mutation tests. Furthermore, as L-arginine is a ubiquitously occurring substance in food, the environment and even in human body fluids there is no concern with respect to mutagenicity. By means of read across it is concluded that L-arginine-HCl should not be classified as a mutagen (as a germ cell mutagen in the wording of CLP).