Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Long history of human exposure to dairy products including yoghurt, without signs of toxicity, teratogenicity or carcinogenicity.

- Chronic 400 mg/kg BW ingestion of Yogurt is considered safe for humans.

- Three in vitro genetic toxicity studies regarding hydrolized milk protein and fermented milk

1) The Mutagenicity of Hydrolyzed Milk Protein was investigated in S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537), with and without metabolic activation. There were no signs of reverse mutations in the used concentrations (up to 5000 µg/plate). Therefore Hydrolyzed Milk Protein was classified as not mutagenic.

2) Mutagenicity of a Hydrolyzed Casein product (MW = 600 Da, 30% solution in water) was investigated in S. typhimurium(TA 98, TA 100, TA 1535, TA 1537) andE. coli (WP2uvrA), with and without metabolic activation. There were no signs of reverse mutations in the used concentrations (up to 5000 µg/plate). Therefore Hydrolyzed Casein was classified as not mutagenic.

3) Clastogenicity of Fermented milk powder: it did not show citotoxic or clastogenic activity on cultured Chinese lung cells, with and without metabolic activation after 6, 24 and 48 h exposure. Therefore Fermented milk powder was classified as not clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Justification for type of information:

Milk proteins are the major component (30-40%) of Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried. The study provided is about hydrolized milk proteins, which are similar to normal milk proteins.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

The complete study is not available. The adherence to GLP is not reported.

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Hydrolized milk protein

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

up to 5000 µg/plate

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Hydrolyzed Milk Protein did not induce reverse mutations, neither with or without metabolic activation by S9 mix. Thus, no indications exist for Hydrolyzed Milk Protein to have potential for gene mutations.

Executive summary:

The Mutagenicity of Hydrolyzed Milk Protein was investigated in S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537), with and without metabolic activation. There were no signs of reverse mutations in the used concentrations (up to 5000 µg/plate). Therefore Hydrolyzed Milk Protein has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Justification for type of information:

Milk proteins are the major component (30-40%) of Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried. The study provided is about hydrolyzed casein, which is a well-known hydrolized milk protein.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

The complete study is not available. The adherence to GLP is not reported.

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Hydrolyzed Casein product (MW = 600 Da, 30% solution in water)

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:

10 % S9 mix

Test concentrations with justification for top dose:

up to 5000 µg/plate

Vehicle / solvent:

Water

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Hydrolyzed Casein did not induce reverse mutations, neither with or without metabolic activation by S9 mix. Thus, no indications exist for Hydrolyzed Casein to have potential for gene mutations on the bacterial strains tested.

Executive summary:

The Mutagenicity of a Hydrolyzed Casein product (MW = 600 Da, 30% solution in water) was investigated in S. typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli (WP2uvrA), with and without metabolic activation. There were no signs of reverse mutations in the used concentrations (up to 5000 µg/plate). Therefore Hydrolyzed Casein has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, near guideline, published in peer reviewed literature, adequate for assessment

Justification for type of information:

The test material used to test the toxicological properties described in the study used for this RSS is not exactly the same as Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried. However, the process used to prepare ‘Powdered Lactobacillus helveticus-fermented milk’ (FM) is qualitatively similar to that used in the production of ‘Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried’ and is based on the same natural material (cow milk) and biological production process (bacterial fermentation). The components that are additionally removed from FM, lactic acid and casein, occur naturally in milk and milk that has been naturally exposed to microorganisms and are not expected to have important toxicological effects regarding acute oral toxicity, repeated dose oral toxicity, reproduction, or genotoxicity at the concentrations present in fermented milk products. Therefore, the toxicological properties reported on this study can be used in combination with the long historical safe consumption of yogurt and yogurt-derived products by humans in a weight of evidence analysis to assess the toxicological properties of 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried' for this endpoint.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Reconstituted skim milk (9%, w/w) was pasteurized and fermented with L. helveticus CM4 at 37°C for 22 h. Casein was removed by centrifugation and lactic acid was eliminated from the supernatant by electrodialysis. The residual supernatant was converted to fermented milk whey powder by using maltodextrin as a bulking agent and spray drying.

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Hygienic Sciences (Tokyo, Japan).
- Methods for maintenance in cell culture: stored in liquid nitrogen
The absence of mycoplasma was confirmed

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver Homogenate (S9) Mix

Test concentrations with justification for top dose:

1250, 2500, or 5000 µg/ml

Vehicle / solvent:

physiological saline

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 6 h
- Rinsing: 3 times with fresh medium
- Recovery incubation: 18 h
Two other group of cells were exposed contiuosly during 24 o 48 h without rinsing and recovery incubation.

SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.1 to 0.2 µg/ml, 2 h before termination of exposure

STAIN (for cytogenetic assays): 3% Giemsa’s solution

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After completion of the incubation period, cells were treated with 0.25% trypsin and harvested by centrifugation (1000 rpm for 5 min). The supernatant was discarded and the resulting pellets were subjected to hypotonic treatment with 0.057 mol/L KCl. The cells were then fixed in cooled Caroy’s solution (methanol–glacial acetic acid).
Two specimens of cells suspended in the Caroy’s solution were placed, side-by-side, on a glass slide and permitted to dry. Dried cells were stained with 3% Giemsa’s solution, washed with water, and dried again.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphases/dose (100 per specimen)

DETERMINATION OF CYTOTOXICITY
- Method: cell survival and proliferation

Evaluation criteria:

Observed structural aberrations were classified as (1) chromatid breaks, (2) chromatid exchanges, (3) chromosome breaks, (4) chromosome exchanges (dicentric, ring, etc.), and (5) fragmentation

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

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:

The results of the cell growth inhibition tests conducted on FM yielded no evidence of treatment-associated cytotoxicity or cell growth inhibition. Degrees of cell confluence in the presence of either test substance were consistently within 85% of that observed during negative control incubations.
Neither short-term nor continuous exposure to FM caused the induction of significant changes in cell growth indices, incidences of chromosomal aberrations or the incidence of polyploids.
Exposures of cells to mitomycin C and benzo(a)pyrene resulted in the induction of aberrant cells after either short-term or continuous exposures consistent with previous laboratory studies using this assay.

Conclusions:

Fermented milk powder did not show citotoxic or clastogenic activity on cultured Chinese lung cells, with and without metabolic activation after 6, 24 and 48 h exposure.
According to the considerations previously listed on the field "justification for type of information", it can be concluded that "Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried" (EC No. 917-734-0) would likewise not show citotoxic or clastogenic activity on cultured Chinese lung cells, with and without metabolic activation after 6, 24 and 48 h exposure.

Executive summary:

Chinese hamster lung (CHL) cells were cultured and exposed in the presence or absence of a rat hepatic metabolizing system (S9) to powdered Lactobacillus helveticus–fermented milk (FM) (1250, 2500, 5000 µg/ml of incubation medium); or positive-control agents, mitomycin C (0.1 or 0.05 µg/ml) or benzo(a)pyrene (20 µg/ml). The cells were incubated for 6 h (either in the presence or absence of S9) or for 24 or 48 h (without S9). Neither short-term nor continuous exposure to either CH or FM caused the induction of significant changes in cell growth indices, incidences of chromosomal aberrations or the incidence of polyploids. Exposures of cells to mitomycin C and benzo(a)pyrene consistently resulted in the induction of the anticipated aberrant cells after either short-term or continuous exposures. The results of these assays support the conclusions that, under the conditions of these experiments, FM does not possesses demonstrable potential for the induction of cytotoxicity or clastogenesis.

The process used to prepare ‘Powdered Lactobacillus helveticus-fermented milk’ (FM) is qualitatively similar to that used in the production of ‘Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried’ and is based on the same natural material (cow milk) and biological production process (bacterial fermentation).The components that are additionally removed from FM, lactic acid and casein, occur naturally in milk and milk that has been naturally exposed to microorganisms and are not expected to have important toxicological effects regarding acute oral toxicity, repeated oral dose toxicity,

reproduction, or genotoxicity at the concentrations present in fermented milk products.

Therefore, taking into account the previous considerations, it is safe to assume that under the conditions of these experiments, 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray dried' would also lack potential for the induction of cytotoxicity or clastogenesis.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are numerous studies on the antimutagenic properties of fermented milk products.

References:

- Fermented milk and milk products as functional foods--a review, 2013, ShinyVK and Mishra HN.

Crit Rev Food Sci Nutr.2013;53(5):482-96. doi: 10.1080/10408398.2010.547398. (and references thereon)

- Safety Assessment of Bovine Milk Proteins and Protein Derivatives as Used in Cosmetics, 2017, Burnett, CL and Heldreth B, Cosmetic Ingredient Review, cir-safety.org. (and references thereon)

- Effects of Milk and Milk Products Consumption on Cancer: A Review, 2013, H Davoodi, S Esmaeili, and AM Mortazavian, Comprehensive Reviews in Food Science and Food Safety, Vol 12, pp 249-264 (and references thereon)

Justification for classification or non-classification

There is a long history of human exposure to dairy products including yoghurt, without signs of chronic toxicity, teratogenicity or carcinogenicity.

Numerous studies have also been done on the antimutagenic properties of milk fermented products.

Milk proteins are the major component (30-40%) of Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried. Two of the in vitro studies provided are about hydrolized milk proteins, which are similar to normal milk proteins.

On a third in vitro study, the clastogenic potential of Fermented milk was assessed. The test material used on this test is not exactly the same as 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried'. However, the process used to prepare ‘Powdered Lactobacillus helveticus-fermented milk’ (FM) is qualitatively similar to that used in the production of ‘Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried’ and is based on the same natural material (cow milk) and biological production process (bacterial fermentation). The components that are additionally removed from FM, lactic acid and casein, occur naturally in milk and milk that has been naturally exposed to microorganisms and are not expected to have important toxicological effects regarding acute oral toxicity, repeated oral dose toxicity, reproduction, or genotoxicity at the concentrations present in fermented milk products. Therefore, the toxicological properties reported on this study can be used in combination with the long historical safe-use of yogurt and yogurt-derived products by humans on a weight of evidence analysis to assess the toxicological properties of 'Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried' for this endpoint.

According to the results obtained with these three in vitro gen toxicity tests for hydrolyzed milk proteins and fermented milk, the history of safe consumption of yoghurt, and the evidence provided by numerous studies on the antimutagenic properties of fermented milk products, Bos taurus, milk, pasteurized, homogenized, skimmed, fermented, spray-dried (EC 917-734-0), is not considered to be genotoxic and there is no need for further testing for genetic toxicity.