Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Comission Directive 2002/32/EC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

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:
Commercially available post-mitocondiral fraction S9 from livers of rodents treated with th eenzyme inducing agent Aroclor
Test concentrations with justification for top dose:
5.00 , 3.00, 2.00, 1.00 and 0.20 mg/plate
Vehicle / solvent:
milliQ water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoantrhacene (S9+, with metabolic activation)
Details on test system and experimental conditions:
The bacterial strains used for the study were grown from controlled Working Banks obtained from Master
Banks (generated in Vivotecnia) in nutrient broth supplemented with the corresponding antibiotics when
required, as follows:
TA98
Nutrient Broth #2 25g/L
Ampicillin 0.025mg/mL

TA100
Nutrient Broth #2 25g/L
Ampicillin 0.025mg/mL

WP2 (pKMl 01 )
Nutrient Broth #2 25g/L
Ampicilin -

TA1535
Nutrient Broth #2 25g/L
Ampicilin -

TA1537
Nutrient Broth #2 25g/L
Ampicilin -

Inoculums were liquid grown overnight up to the late exponential-early stationary phase of growth
(approximately 1.2-1.4 OD at 660nm). This OD indicates that bacteria are growing in the late exponential or
early stationary phase of growth (approximately 1-2x10E9 CFU/mL).

The following types of agar medium were used in the test:
Media Agar Glucose Vogel-Bonner NaCI Histidine Biotin Tryptophan
Minimal agar 1.5% w/v 0.2% w/v 2% v/v - - - -
Top agar Salmonella 0.54% w/v - - 0.45% w/v 0.05mM 0.05mM -
Top agar E.Coli 0.54% w/v - - 0.45% w/v - - 0.05mM

Evaluation criteria:
The criteria used for determining a positive result take into account a dose-response effect in the range
tested andlor a reproducible increase at one or more concentrations in the number of revertant colonies per
plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item treated plates is increased
when compared to the solvent treated plates according to the following criteria:
species strain criteria
S.typhimurium TA98 2 fold
S.typhimurium TA100 2 fold
S. typhimurium TA102 2 fold
S.typhimurium TA1535 3 fold
S.typhimurium TA1537 3 fold
E.coli WM'2(pKM101 ) 2 fold

Biological relevance of the results was also considered.

Results and discussion

Test resultsopen allclose all
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:
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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
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
Additional information on results:
The number of revertant colonies per plate was counted and recorded by an automatic colony counter.
Average plate counts was presented with the mean and the standard deviation for each set of triplicates per
test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the
corresponding negative control.

Colony counting evaluation (R value)
None of the concentrations assayed for the test item showed an increase in the R value either with or without
S9 metabolic activation regardless of the procedure.
No growih was observed in the bacteria strain E. coli WP2 without metabolic activation system under
preincubation procedure.
This condition and E. coli WP2 without metabolic activation system under direct incorporation procedure
were repeated in order to confirm the obtained results.
The decrease in the R value of the bacteria strain E. coli WP2 without metabolic activation system under
preincubation procedure was alsoobserved in the repetition.

Dose response evaluation
No dose response for the test item Ac-Gly-beta-Ala-OH was observed in any of the tested bacterial strains.

Applicant's summary and conclusion

Conclusions:
The substance showed no mutagenic effects in the performed Bacterial Reverse Mutation test (OECD 471), both with and without metabolic activation.
Therefore, the substance is considered to be non mutagenic and non promutagenic.
Executive summary:

The bacterial reverse mutation test (Ames test) assesses the mutagenic or promutagenic potential of the test

item Acetyl glycyl beta.alanine in the several bacterial strains.

The test was performed in accordance with OECD Guideline 471 for the Testing of Chemicals (Bacterial

Reverse Mutation Test. Adopted 21st July 1997) and the test Method B13/B14 of Commission Directive

2000/32/EC.

No cytotoxic activity was observed at a test item concentration of 50.0mg/mL.

Five test item doses ranged between 5.00 and 0.2 mg/plate were assayed. None of the concentrations

assayed for the test item showed an increase in the R value either with or without S9 metabolic activation

regardless of the procedure.

No dose response for the test item acetyl glycil beta-alanine was observed in any of the tested bacterial strains.

Based on the results obtained in this study, it can be concluded that the test item does not induce point

mutations or frame-shifts in the genome of the bacterial strains with or without metabolic activation

regardless of the procedure.

Therefore, the test item Acetyl glycyl beta-alanine is considered to be NON MUTAGENIC / NON PROMUTAGENIC

under the experimental conditions assayed.