L-(+)-2,5-diaminopentanoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Genotoxicity Studies of Drugs and the Standards for Mutagenicity Tests Using Microorganisms

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

L-(+)-2,5-diaminopentanoic acid

EC Number:

221-678-6

EC Name:

L-(+)-2,5-diaminopentanoic acid

Cas Number:

3184-13-2

Molecular formula:

C5H12N2O2.ClH

IUPAC Name:

L-(+)-2,5-diaminopentanoic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kyowa Hakko Bio Co., Ltd. Batches 080926 and 083019
- Expiration date of the lot/batch: practically stable
- Purity test date: 1993-08-27
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stable
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: highly soluble
-
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
Purity >/= 99.9 %

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test: 13, 26, 53, 105, 211, 422, 843, or 1686 µg/mL
Justification: Guidelines

Short-term exposure experiments: 422, 843, or 1686 µg/mL.
Justification: 1686 µg/mLnegative in preliminary test.

Continuous experiments: 422, 843, and 1686 µg/mL
Justification: 1686 µg/mLnegative in preliminary test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

A preliminary cell growth inhibition test was conducted in order to select the concentrations of L-ornithine monohydrochloride to be used in the chromosome aberration test. In accordance with the Japanese Ministry of Labor guidelines, the top dose tested in the range finding assay was 0.01 M (equivalent to 1686 µg/mL). Briefly, 2 x 10exp4 cells were seeded into a 60 mm culture dish and incubated for 3 days. The test article was applied to the culture medium at concentrations of 0 (solvent control) 13, 26, 53, 105, 211, 422, 843, or 1686 µg/mL and cells were incubated for either 24 or 48 h at 37 °C. The procedure was repeated with the addition of S9 mix. Cell growth inhibition rate was measured as a percentage of cells in the solvent control.

In the short-term exposure experiments, L-ornithine was dissolved in physiological saline and added to the culture media at final concentrations of 422, 843, or 1686 µg/mL, in the presence or absence of exogenous metabolic activation (S9 mix). Untreated cultures and cultures incubated with saline were used as the negative controls, and 0.10 µg/mL mitomycin C (absence of exogenous metabolic activation) or 7.0 lg/mL benzo[a]pyrene (presence of exogenous metabolic activation) were used as positive controls. Cells were incubated with the solution containing the test article or controls for 6 h at 37 °C, followed by a rinse and further incubation in untreated culture media for an additional 18 h at 37 °C (recovery period). Colcemid (0.2 µg/mL) was added to the dishes 2 h before the end of the recovery period.

In the continuous experiments, cells were incubated with the solution containing the test article at final concentrations of 422, 843, and 1686 µg/mL for 24 or 48 h in the absence of metabolic activation. Untreated cultures and cultures incubated with saline were used as negative controls, and 0.05 µg/mL of mitomycin C was used as a positive control. Colcemid (0.2 µg/mL) was added to the dishes 2 h prior to the end of the treatment period. All cultures were prepared in duplicate.

Evaluation criteria:

The frequency of chromosome aberrations or polyploidy did not exceed 5% at any of the concentrations of L-ornithine monohydrochloride tested, in either the short-term or continuous experiments. The frequency of chromosome aberrations in cells treated with mitomycin C or benzo[a]pyrene were consistent with historical positive control data.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the conditions of the experiment, it was concluded that L-(+)-2,5-diaminopentanoic acid did not induce chromosome aberrations or polyploidy.

Executive summary:

L-(+)-2,5-diaminopentanoic acid was tested for genetic toxicity in an in vitro cytogenicity study in mammalian cells.

No evidence of genotoxicity was observed in the chromosome aberration test at doses of up to 1686 mg/mL, both in the presence and absence of metabolic activation.