Fytic acid

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:

15.11.2011 -25.11.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997
“Bacterial Reverse Mutation Test“

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EU-Guideline B.13/14 adopted 31. May 2008 “Mutagenicity –Reverse mutation test
using bacteria

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

myo-Inositol, hexakis(hydrogen phosphate), hexasodium salt (all isomers)

Molecular formula:

NA6C6H12O24P6

IUPAC Name:

myo-Inositol, hexakis(hydrogen phosphate), hexasodium salt (all isomers)

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Aqua; Water

additive 1

Reference substance name:

Ethanol

EC Number:

200-578-6

EC Name:

Ethanol

Cas Number:

64-17-5

Molecular formula:

C2H6O

IUPAC Name:

Ethanol

Test material form:

liquid

Details on test material:

- State of aggregation: Liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

1. Experiment: 50µg, 150 µg, 500 µg, 1499 µg, 4995 µg/ plate
2. Experiment: 314µg, 627 µg, 1254 µg, 2507 µg, 5013 µg/ plate
The highest dose was selected on the basis of the preliminary cytotoxicity test

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO; Water
- Justification for choice of solvent/vehicle: Based on solubility of selected substances
Justification for choice of solvent/vehicle: DMSO/Water
DMSO was a suitable vehicle for exposure to the test material up to the maximum guideline recommended test material concentration of 5000 μg/plate. It was compatible with bacterial survival and S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

used as negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2 - Amino-anthracene CAS 613-13-8 1 µg/plate Solvent: DMSO, with metabolic activation S9; 4-Nitro-1,2-phenylene diamine CAS 99-56-9; 20 µg/plate; solvent: DMSO, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I:
In agar (plate incorporation),
Experiment II
Pre - incubation method (opreincubation period at 37°C: 20 minutes)

DETERMINATION OF TITRE:
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation over night at 37°C followed. It should give a density of 109 cells/mL (at the least).

DURATION
- Exposure duration: 48h/ 37°C

NUMBER OF REPLICATIONS: 4

DETERMINATION OF CYTOTOXICITY
- Toxicity was tested with all strains at one concentration 4995 µg/plate, plate incorporation method, replicants: 4
- Any supplementary information relevant to cytotoxicity:

Negative Control:
Solvents DMSO and H2O were tested , replicants 4, all strains, with and without metabolic activation

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous
revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ³ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
All calculations are performed with unrounded values.

Results and discussion

Test results

Test results 1

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

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 97 a

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

Test results 3

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

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:

valid

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:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item is considered as “not mutagenic under the conditions of the test”.

Executive summary:

The aim of this test was to determine the mutagenic potential of the test item, following the test design given in OECD 471.

The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.” The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity.

Two valid experiments were performed.

First Experiment:

Five concentrations of the test item, dissolved in deionised water (ranging from 4995 to 50 μg/ plate) were used. Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn’t show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 5013 to 314 μg/ plate) and a modification in study performance (pre-incubation method). The test item didn’t show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Under the conditions of the test, the test item didn’t show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. Therefore, no concentration-effect relationship could be determined.

The test item is considered as “not mutagenic under the conditions of the test”.