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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
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:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: In vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
The tested substance is a protein hydrolysate from the GlycoMacroPeptide (GMP) fraction which is derived from cow’s milk. The processed
final product, a supplement or a functional food ingredient, contains high levels of oligopeptides, especially the tripeptide Isoleucine-Proline-Proline (IPP)
The tested substance is known with the commercial name TENSGUARD

Method

Target gene:
human lymphocytes
Species / strain
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The test concentrations ranged from 1250 to 5000 ug/mL
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
The chromosomal aberration test in cultured human lymphocytes was performed in compliance with OECD guideline no. 473. The tested TensguardTM concentrations ranged from 1250 to 5000 lg/ml. Cells treated for the pulse exposure (4 h treatment) were exposed in the absence and presence of S9-mix, cells treated for the continuous exposure (24 h treatment) were exposed only in the absence of the S9-mix. The final concentration of liver homogenate fraction was 4%. Negative controls (i.e. solvent DMSO) and positive controls (mitomycin C and cyclophosphamide in the absence and presence of the S9-mix respectively) were run simultaneously.
At least 1000 stimulated lymphocytes (500 on each slide) were examined in each culture to determine the percentage of cells in mitosis (mitotic index). On the basis of the results of the mitotic index scoring and the observations with respect to the quality of the metaphases, three concentrations of TensguardTM together with the negative and positive controls were selected for chromosomal aberration analysis. If possible, the highest concentration should reduce the mitotic index by at least 50% (but not more than 70%), when compared to the negative control value or exhibit some other clear indication of cytotoxicity. Since the mitotic index was not reduced by more than 50% at any concentration of TensguardTM tested, three concentrations up to the maximum of 5000 lg/ml were selected for the chromosomal aberration test. Subsequently, the cultures of the selected concentrations of TensguardTM, together with the negative and positive control cultures, were analyzed for structural chromosomal aberrations. The assay was considered valid if the positive controls showed a statistically significant increase (p < 0.05) in the number of aberrant cells and if the negative controls were within the historical range. The test substance was considered to be clastogenic if a concentration-related increase in the percentage of cells with structural aberrations over the concurrent control frequencies was observed, or if a statistically significant and reproducible increase was observed at a single concentration. Cells with only gaps, multiple aberrations, polyploidy and endoreduplication were recorded separately and not included in the final assessment of clastogenic activity.
Rationale for test conditions:
See previous explanation (Detalis on test system and conditions)
Evaluation criteria:
See previous explanation (Detalis on test system and conditions)
Statistics:
The assay was considered valid if the positive controls showed a statistically significant increase (p < 0.05) in the number of aberrant cells and if the negative controls were within the historical range

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
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:
The study shows that the tested hydrolysate protein is not genotoxic according to guideline OECD 473