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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Only references to studies are reportred in the attached documents
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test material is casein hydrolysate
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Up to 5000 ug/plate
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Conclusions:
Hydrolyzed Casein The mutagenic potential of a Hydrolyzed Casein product (MW = 600 Da, 30% solution in water) was studied in an Ames test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 and Escherichia coli strain WP2uvrA, with and without S9 metabolic activation.17 Concentrations were tested up to 5000 μg/plate. The test material did not induce reverse mutations with or without S9. It was concluded that Hydrolyzed Casein, Hydrolyzed Keratin and Hydrolyzed Silk are not mutagenic.

Hydrolyzed Milk Protein
Distributed for comment only -- do not cite or quote

The potential of Hydrolyzed Milk Protein to induce gene mutation was studied in S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation.38 Concentrations were tested up to 5000 μg/plate. The test material did not induce reverse mutations with or without S9. It was concluded that Hydrolyzed Milk Protein was not mutagenic.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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
Target gene:
Salmonella typhimurium
Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The test concentrations ranges from 62 to 5000 ug/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: N-ethyl-N-notrosourea and 2-aminoanthracene
Details on test system and experimental conditions:
The bacterial reverse mutation test was performed in compliance with OECD guideline no. 471 using the plate incorporation method with the histidine-requiring Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the tryptophanrequiring Escherichia coli strain WP2 uvrA in the absence and presence of a liver fraction of Arochlor 1254-induced rats for metabolic activation (S9-mix). The final concentration of liver homogenate fraction was 10%. Five TensguardTM concentrations were used ranging from 62 to 5000 lg/plate. Negative controls (i.e. the solvent
milliQ water) and positive controls were run simultaneously. The positive control substances were sodium azide (TA1535 and TA100), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98) and N-ethyl-N-nitrosourea (WP2 uvrA) in the absence of S9-mix and 2-aminoanthracene (TA1535, TA98, TA 100 and WP2 uvrA) and benzo(a)pyrene (TA1537) in the presence of the S9-mix. Bacteria were exposed to the substances at 37 C, for approximately 72 h. Toxicity was defined as a reduction (at least 50%) in the number of revertant colonies and/or clearing of the background lawn of bacterial growth. The assay was considered valid if the mean colony counts of the control values of the strains were within acceptable ranges and if the results of the positive controls met the criteria for a positive response (i.e. a two-fold increase compared to the negative control). The test substance was considered to be mutagenic if the mean number of revertant colonies on the test plates was increased in a concentration-related way or if a reproducible two-fold or more increase was observed compared to that of the negative control plates.
Rationale for test conditions:
See prevoius box
Evaluation criteria:
See prevoius box
Statistics:
See prevoius box
Key result
Species / strain:
S. typhimurium TA 1535
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

In the bacterial reverse mutation test, TensguardTM was not mutagenic as evidenced by the absence of a dose-related or a more than a two-fold increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed for the negative control. Furthermore, TensguardTM was not cytotoxic to any strain. The positive control substances gave the expected increase in the number of revertant showing the validity of the test

Conclusions:
The study shows that the tested hydrolysate protein is not genotoxic according to guideline OECD 471
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: In vitro mammalian chromosome aberration test
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
Target gene:
human lymphocytes
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
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
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
Conclusions:
The study shows that the tested hydrolysate protein is not genotoxic according to guideline OECD 473
Endpoint:
in vitro gene mutation 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
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
Target gene:
LY5178Y cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The test concentration range from 310 to 5000 ug/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
The gene mutation test in LY5178Y cells was performed in compliance with OECD guideline no. 476 using the microtiter-plate method. Duplicate cultures of mouse lymphoma L5178Y cells were exposed for 4 h in the presence of S9-mix and 24 h in the absence of S9-mix. The final concentration of liver homogenate fraction was 2%. Five TensguardTM concentrations were tested ranging from 310 to 5000 lg/ml. Negative controls (i.e. culture medium without serum) and positive controls (methyl methanesulphonate and 3-methylcholanthrene in the absence and presence of the S9-mix respectively) were run simultaneously. After treatment, cells were grown for 48 h to allow mutation fixation and, subsequently, cells were seeded on microtiter-plates in the absence and presence of trifluorothymidine (TFT). To assess cytotoxic effects, the initial cell yield after treatment, the suspension growth and the total growth (during 48 h culturing after treatment) relative to that of the solvent negative controls were calculated. The mutant frequency was expressed as the number of TFT-resistant mutants per 1,000,000 clonable cells.
A response was considered positive if the mutant frequency was increased by more than 100 mutants per 1,000,000 clonable cells. Any apparent increase in mutant frequency
at concentrations of the test substance causing more than 90% cytotoxicity was considered to be not indicative of genotoxicity. The test substance was considered to be mutagenic if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
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:
See previous explanation (Detalis on test system and conditions)
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The gene mutation test in mouse lymphoma LY5178Y cells showed that TensguardTM did not increase the mutation frequency at the TK-locus at any dose level in the absence and presence of S9-mix. In addition, TensguardTM was not cytotoxic to the L5178Y cells and the relative total growth at the highest concentration tested was not decreased compared to the negative control. The positive control substances yielded the expected significant increase in mutant frequency compared to the negative controls which were within acceptable ranges.

A test following OECD471 guideline was performed as well with the same substance, giving a negative result

Conclusions:
The study shows that the tested hydrolysate protein is not genotoxic according to guidelines OECD 476 and 471
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
not specified
Remarks:
In the article is not specified if the thest was performed under GLP compilance
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
S. horrens protein hydrolysate was supplied by Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia. All other reagents used in this study were of analytical grade
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Test concentrations with justification for top dose:
12.5 ug/mL
25 ug/mL
50 ug/mL
Vehicle / solvent:
DMEM
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
In vitro micronucleus test was conducted for genotoxicity evaluation of the S. horrens hydrolysates sample, as per OECD guidelines number 487. Chinese hamster lung fibroblast cells (V79 cells) were cultured under standard conditions in DMEM supplemented with 10% heat-inactivated-FBS, 0.2 mg/ml L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were kept in tissue-culture flasks at 37ºC in a humidified atmosphere, containing 5% CO2 in air, and were harvested by treatment with 0.15% trypsin–0.08% EDTA in phosphate-buffered saline solution (PBS). The cell culture then was treated with S. horrens hydrolysates with concentration of 12.5 μg/ml, 25 μg/ml and 50 μg/ml and incubated. DMSO was used as negative control and clastogenic agent mitomycin C as positive control. The cells were observed under fluorescence microscope after stained with acridine orange dye for the formation of micronuclei.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The study performed with S. horrens hydrolysate protein following OECD gudelines 471 and 487 shows that the substance is not genotoxic
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification