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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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'.

Applicant's summary and conclusion

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.