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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:
2015-11-17 to 2015-11-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix
EC Number:
701-304-2
Cas Number:
1353091-50-5
Molecular formula:
(SiO2)(Fe2O3)
IUPAC Name:
High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix
Test material form:
solid: particulate/powder
Details on test material:
- Name: Colorante Rojo
- OLD EC Name: Reaction mass of fumes, silica and diiron trioxide
- NEW EC Name: High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix
- Substance type: inorganic pigment
- Physical state: solid, red powder, odourless
- Storage condition of test material: at room temperature

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment II: 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation.
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:
Positive control (without metabolic activation): concentration: 10 μg/plate (strains TA 1535 & TA 100)
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:
Positive control (without metabolic activation): concentration: 10 μg/plate (strain TA 98) & 50 µg/plate (strain TA 1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control (without metabolic activation): concentration: 2.0 μL/plate (strain E.coli WP2 uvrA)
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:
Positive control (with metabolic activation): concentration: 2.5 μg/plate (strains TA1535, TA1537, TA98 & TA100) & 10 µg/plate (strain E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-experiment/Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration (Experiment I & II): at least 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. This pre-experiment was conducted as plate incorporation test.
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA98, TA100, and WP2 uvrA) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Evaluation criteria:
A test item is 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 is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not mandatory.

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
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 μg/plate. The undissolved particles had no influence on the data recording.

DOSE SELECTION:
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 5000 μg/plate were chosen as maximal concentration. Based on the observed precipitation the second experiment was performed with 7 concentrations.

CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

EXPERIMENT I & EXPERIMENT II:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. A minor increase below the threshold of two was observed in Experiment II in strain TA98 in the absence of S9 mix at 2500 μg/plate (1.7). Since the values are within the range of the historical control data of the solvent control it is judges as biologically irrelevant.

Please also refer for results to the field "Attached background material" below.

Applicant's summary and conclusion

Conclusions:
The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.