Protein hydrolyzates, milk

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Protein hydrolyzates, milk

EC Number:

296-575-2

EC Name:

Protein hydrolyzates, milk

Cas Number:

92797-39-2

IUPAC Name:

Protein hydrolyzates, milk

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 Ng/plate. The
pre-experiment is reported as experiment I. Since no toxic effects were observed
5000 Ng/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations
were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 Ng/plate
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Ng/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 Ng/plate
The plates incubated with the test item showed normal background growth up to
5000 Ng/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants (below the

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

Remarks:

With S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

The histidine dependent strains are derived from Salmonella typhimurium strain LT2
through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus)
mutation they possess a faulty lipopolysaccharide envelope which enables substances to
penetrate the cell wall more easily. A further mutation causes a reduction in the activity of
an excision repair system. The latter alteration includes mutational processes in the nitrate
reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus".
In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin
resistance marker (6).
Strain WP2 (4) and its derivatives all carry the same defect in one of the genes for
tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise
either by a base change at the site of the original alteration or by a base change
elsewhere in the chromosome so that the original defect is suppressed. This second
possibility can occur in several different ways so that the system seems capable of
detecting all types of mutagen which substitute one base for another. Additionally, the
uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a
repair-deficient strain may be more readily mutated by agents.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

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

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 4

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

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

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the
experimental conditions reported, the test item did not induce gene mutations by base pair
changes or frameshifts in the genome of the strains used.
Therefore, YOGURTENE 20 CQ (PFR) U/A - 6099943 is considered to be non-mutagenic
in this Salmonella typhimurium and Escherichia coli reverse mutation assay.