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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
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. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
additive
Test material form:
liquid
Details on test material:
- State of aggregation: Liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
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 resultsopen allclose all
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
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
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:
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”.