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Toxicological information

Genetic toxicity: in vitro

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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:
Between 25 July 2012 and 19 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lanthanum acetate
- Substance type: White solid
- Physical state: Solid
- Storage condition of test material: Room temperature in the dark over silica gel under nitrogen

Method

Target gene:
Salmonella typhimurium
TA1537: his C 3076; rfa-; uvrB- (frame shift mutations)
TA98: his D 3052; rfa-; uvrB-; R-factor (frame shift mutations)
TA1535: his G 46; rfa-; uvrB- (base-pair mutations)
TA100: his G 46; rfa-; uvrB-; R-factor (base-pair mutations)

Escherichia coli
WP2 uvrA: trp-; uvrA- (base-pair substitution)
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:
S9 microsomal fraction from rats induced with phenobarbitone/beta-naphtoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (lanthanum acetate as active ingredient)
Mutation test - experiment 1:
50, 150, 500, 1500, 5000 µg/plate (lanthanum acetate as active ingredient)
Mutation test - experiment 2:
5, 15, 50, 150, 500, 1500, 5000 µg/plate (lanthanum acetate as active ingredient)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535; without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537; without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
0.2 µg/plate for TA98; without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA; with S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98; with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation method

DURATION
- Preincubation period: 20 minutes (mutation test - experiment 2 - pre-incubation method)
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): simultaneous with exposure

SELECTION AGENT (mutation assays): trace histidine or tryptophan supplemented

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertant colonies and reduction of bacterial background lawn
- Preliminary toxicity test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (lanthanum acetate as active ingredient). The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test item formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 mL of the maximum concentration of the test item and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.

OTHER: S9 - mix was used at 10% in plates incubated with metabolic activation
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989))
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response.
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

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:
cytotoxicity
Remarks:
A visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both with and without S9-mix in both experiments.
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:
cytotoxicity
Remarks:
A visible reduction in the growth of the bacterial background lawns of WP2 uvr A at 5000 µg/plate in both with and without S9-mix in both experiments.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutation test:
The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix employing plate incorporation and pre-incubation methodologies. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was noted at 5000 µg/plate in both the absence and presence of S9-mix in Experiments 1 and 2, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item, lanthanum acetate, was considered to be non-mutagenic under the conditions of this test.