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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The reliability of the source read-across study was established to be R2: published study well documented
Justification for type of information:
Justification for read-across is detailed at section 13.

Data source

Reference
Reference Type:
publication
Title:
Effects of amino acids on sister-chromatid exchanges.
Author:
Zhang and Yang
Year:
1992
Bibliographic source:
Mutation Research, 280 (1992) 279-283

Materials and methods

Principles of method if other than guideline:
Sister chromatid exchange Human peripheral blood Lymphocytes.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Lysine
CAS no.: 56-87-1
EC no.: 200-294-2

Method

Species / strain
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 10, 50 and 100 µg/ml
Vehicle / solvent:
Saline solution
Controls
Untreated negative controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION
The whole-blood microculture technique was used. 0.4 ml of venous whole blood was inoculated to 5 ml of the culture medium.

CULTURE MEDIUM
- Composition: containing
41.6 mg of RPMI 1640 (JR Scientific Inc., Woodland, CA)
1 ml of fetal calf serum (purchased from Tianjin Medical College, China)
1.5 mg of PHA (produced by Guangdong Biological Product Institute, China)
10000 IU each of penicillin and streptomycin
0.1 ml of 5 % NaHCO3.
- pH: 7

INCUBATION
The mixture was incubated for 24 h. Bromodeoxyuridine (BrdU), at 10 µg/ml, was added at 24 h of culture.
At 48 h of culture, substance (dissolved in saline solution) or saline solution (for control) was added and the cultures were incubated for another 24 h.

HARVESTING
Peripheral Blood Lymphocytes (PBL) were harvested at the end of the 24 h exposure period (total culture time = 72 h) with 0.05 µg/ml colchicine being added 2 h before harvesting.
All cultures were incubated at 37 °C in the dark.
PBL, harvested after gentle centrifugation (1000 rpm, 10 min) and discarding the supernatant, were resuspended in 0.075 M KCI solution for hypotonic treatment for 20 min, centrifuged again, and finally fixed in methanolacetic acid solution (3:1) three times.

SLIDES PREPARATION
A few drops of the cell suspension were dropped onto a clean slide taken from icy water and frame-dried. The slides were incubated in 2 × SSC solution in petri dishes at 70 °C and irradiated simultaneously under a UV lamp (30 W) at a distance of 10 cm for 20 min. The preparations were stained in 3 % Giemsa solution for 10 min after being rinsed with tap water (Xing, 1990; Zhang, 1991).

SCE ANALYSIS
SCE analysis was carried out on cells having replicated for two cycles in the presence of BrdU. Ten metaphases were scored for each treatment.
Statistics:
Statistical methods for analysis of variance (AOV) based on the square roots of SCE were used (Crossen, 1977; Morgan, 1977, 1981; Vercauteren, 1984)

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Untreated negative controls validity:
valid
Additional information on results:
After multiple comparisons, the effects of test article on SCE in PBL are not significant.

Further substances were involved in the test and divided into two groups with each group consisting of five substances. A randomized complete-block design was used to avoid the influence of individual differences on SCE. One person's blood was used for each test and regarded as one block.
Remarks on result:
other: no mutagenic potential

Any other information on results incl. tables

 

 

Applicant's summary and conclusion

Conclusions:
No mutagenic potential
Executive summary:

Effects of Target substance on SCE in PBL

The experimental data show that the treatment with test substance did not induce a significant increase in SCE. The test item is an essential substance for PBL growth. Thus, it was unexpected that the target substance can significantly induce SCE in PBL. We infer that the addition of exogenous substances might result in an imbalance among tested substances in the medium and cause metabolic disturbances in cells. This might further influence the activities of various enzymes and result in the induction of SCE.

However, this is only a hypothesis and the actual molecular mechanisms involved remain unsolved.