Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-02-08 to 2007-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D.
Version / remarks:
dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): T002675, TIC876
- Physical state: liquid
- Appearance: colorless to yellowish liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 603T-1
- Expiration date of the lot/batch: 2007-12-19
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicqted
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

Method

Target gene:
histidine locus (S. typhimurium strains); tryptophan locus (E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Napthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment/Experiment 1: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9;
Experiment 2: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9;

Since the test item was soluble in deionised water up to the stanard limit concentration recommended in the regulatory guidelines that this assay followed (5000 µg/plate), the highest tested concentration in the Pre-experiment was 5000 µg/plate.
The highest tested concentration in the mutation experiment 2 was selected based on the toxicity of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties; no precipitation of the test substance occurred up to the highest investigated dose.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation; at 10 µg/plate (TA100 and TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation; at 10 µg/plate (TA98), at 50 µg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation; at 3 µL/plate (WP2uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation; at 2.5 µg/plate (TA1535, TA1537, TA98 and TA100); at 10µg/plate (WP2uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I - in agar (plate incorporation);
- Experiment I - plate incorporation:
In the plate incorporation assay, the following materials were mixed in a test tube and poured onto the selective agar plates for each dose level: 100 µL test solution, solvent (negative control) or reference mutagen solution (positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µL bacteria suspension (cf. test system, pre-culture of the strains), and 2000 µL overlay agar (molten at 45 °C)
- Experiment II - preincubation:
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 60 min (experiment II)
- Exposure duration: at least 48 hours (experiments I and II)
- Selection time (if incubation with a selection agent): at least 48 hours (experiments I and II; simultaneous with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): at least 48 hours

SELECTION AGENT (mutation assays): histidine (TA98, TA100, TA1535, TA1537); tryptophan (Wp2uvrA)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.
Rationale for test conditions:
Solubility limitations: Since the test item was fully soluble in deionised water, the highest tested concentration for the Mutation assay was 5000 µg/plate.
Evaluation criteria:
- The test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls, such an increase was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data was not mandatory.
The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both the individual and mean values of the plates for each concentration, together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Water solubility: < 60g/L
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:
- No dose range-finding test was performed. However, in the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I since no toxic effects were observed and 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. Based on the results of experiment I, 5000 µg/plate was selected as the highest test item concentration for experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
- in both experiments, the data in negative control, solvent control and positive controls were within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in both the plate incorporation and pre-incubation experiments. No toxic effects, evident as a substantial reduction in the mean revertant colony counts or by a sparse or absent background bacterial lawn, occurred in the test groups either with or without activation.
Remarks on result:
other: Experiment I and II

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

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, T002675 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.