Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Tara gum
EC Number:
254-409-6
EC Name:
Tara gum
Cas Number:
39300-88-4
IUPAC Name:
Oligosaccharides of Caesalpinia Spinosa extract
Test material form:
liquid
Details on test material:
Aqueous yellow liquid

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
N/A
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of rats
Test concentrations with justification for top dose:
The solubility test showed no insolubility of the test item. Therefore, the maximal concentration retained was 5000 μg/plate.
According to OECD Guideline, 5 concentrations of test item have been studied with approximately halflog (i.e. approximately √10) interval. These doses (rounded to the higher value) used for the preliminary cytotoxicity test were therefore the following: 5000, 1600, 500, 160 and 50 μg/plate.
As the preliminary experiment revealed no cytotoxicity of the test item, this range of concentrations has been conserved for the Test 1.
According to the results obtained in the Test 1, the Study Director decided to maintain range of concentrations for Test 2.
Each test item dilution and each reference item are tested on 3 Petri plates.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test substance was soluble in water.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
water or DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
Preliminary cytotoxicity study
The preliminary cytotoxicity study of the test item has been performed on the strain S. typhimurium TA100, in the same conditions as the Test 1.
Results obtained are part of the Test 1 results if no cytotoxicity is observed. The test item has been dissolved in the suitable solvent.
The applied protocol was the following one:
• In 3 hemolysis tubes, introduce:
o assay without metabolic activation:
 0.5 ml sterile phosphate buffer 0.2 M, pH 7.4,
 0.1 ml of the different test item concentrations,
 2 ml of top agar,
 0.1 ml of bacterial inoculum (TA100).
o assay with metabolic activation:
 0.1 ml of the different test item concentrations,
 2 ml of top agar,
 0.1 ml of bacterial inoculum (TA100),
 0.5 ml of S9-Mix.
• Mix and pour on the surface of the bottom agar previously distributed in Petri dishes.
• Incubate at 37 °C (± 1 °C) for 48 to 72 hours.

Test itself: Research of mutagenic activity
For at least 5 concentrations of the test item, a test without metabolic activation and a test with metabolic activation have been performed simultaneously as follow:
Test 1: direct method
• For each strain, in 3 hemolysis tubes, introduce:
o assay without metabolic activation:
 0.5 ml sterile phosphate buffer 0.2 M, pH 7.4,
 0.1 ml of the different test item concentrations,
 2 ml of top agar

Test 2: method with pre-incubation
Pre-incubation method allows revealing more effectively mutagen activity of some compounds like
aliphatic nitrosamines, bivalent metals, aldehydes, azoic coloring agent.
The applied protocol was the following one:
• For each strain, in 3 hemolysis tubes, introduce:
o assay without metabolic activation:
 0.5 ml sterile phosphate buffer 0.2 M, pH 7.4,
 0.1 ml of the different test item concentrations,
 0.1 ml of bacterial inoculum.
o assay with metabolic activation:
 0.1 ml of the different test item concentrations,
 0.1 ml of bacterial inoculum,
 0.5 ml of S9-Mix.
• Incubate at 37°C (± 1°C) for 20 to 30 min.
• Add the 2 ml of top agar.
• Mix and pour on the surface of the bottom agar previously distributed in Petri dishes.
• Incubate at 37°C (± 1°C) for 48 to 72 hours.

 0.1 ml of bacterial inoculum.
o assay with metabolic activation:
 0.1 ml of the different test item concentrations,
 2 ml of top agar,
 0.1 ml of bacterial inoculum,
 0.5 ml of S9-Mix.
• Mix and pour on the surface of the bottom agar previously distributed in Petri dishes.
• Incubate at 37°C (± 1°C) for 48 to 72 hours.
Rationale for test conditions:
For each assay the following observations were performed and reported:
• Observation of the reagent mix before Petri plates pouring: reporting of any abnormal sign (precipitate, trouble, etc.),
• Petri plates observation and reporting of any cytotoxicity sign (bottom bacterial layer reduction). The cytotoxicity intensity on the bottom bacterial layer is evaluated qualitatively on each plate by naked eyes:
o total destruction of the bottom bacterial layer (the revertants development does not occurs in this case), this one is noted in tables of results as “A”.
o moderated destruction of the bottom bacterial layer. This one is noted in tables of results as “S”.
Acquisition and storage of raw data were managed by the following electronic system:
 Reading of plates: Sorcerer, version 2.2.
 Transfer and storage of raw data: Ames Study Manager, version 1.22.
Evaluation criteria:
The test is considered valid if the following criteria are fulfilled:
 The sterility tests are conform,
 The mean negative controls are within the historical data,
 The solvent used (negative control) must not show genotoxic nor cytotoxic activity,
 The revertants rate obtained for the positive controls must be in agreement with the historical data,
 The positive controls must show a revertants number equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 (R ≥ 2) and the triple of the spontaneous rate of reversion for TA1535 and TA1537 (R ≥ 3),
 No more than 5% of the plates of the test are lost through contamination or any other unforeseen event,
 At least 3 concentrations are available for mutagenicity assessment.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA98, TA100 and TA 102
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:
Revertant analysis tables show that:
 No cytotoxic effect was observed,
 No concentration of the test item showed ratio R higher or equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 strains and to the triple of the spontaneous rate of reversion for TA1535 and TA1537 strains, with and without metabolic activation,
 No dose response was observed, whatever the test system or conditions of the test.
 In addition, no sign of precipitate was observed.

Applicant's summary and conclusion

Conclusions:
Based on the result of this study, the test item was found to be non mutagenic and non pro-mutagenic under the test conditions.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to test item at the following concentrations: 50, 160, 500, 1600 and 5000μg/plate.

Based on the result of this study, the test item was found to be non mutagenic and non pro-mutagenic under the test conditions.