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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:
From 11 October 2012 to 29 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented GLP study performed according to OECD Guideline 471 (adopted July 21, 1997) and EU Guideline B.13/14 (31 May 2008). A deviation of the temperature was observed in the incubation period (39.4°C instead of 37.0°C for approximately 1.5 hour) in the second experiment but the test integrity was not adversely affected.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
A deviation of the temperature was observed in the incubation period (39.4°C instead of 37.0°C for approximately 1.5 hour) in the second experiment but the test integrity was not adversely affected.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
A deviation of the temperature was observed in the incubation period (39.4°C instead of 37.0°C for approximately 1.5 hour) in the second experiment but the test integrity was not adversely affected.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Barium nitrate
- Substance type: white crystals
- Physical state: solid
- Analytical purity: 99.52%
- Lot/batch No.: 120724
- Expiration date of the lot/batch: 07 September 2013
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
The characteristics of the different strains were as follows:
- TA1537: Histidine mutation hisC3076 mutation detecting frameshift
- TA98: Histidine mutation hisD3052/R-factor mutation detecting frameshift
- TA1535: Histidine mutation hisG46 mutation detecting base-pair substitutions
- TA100: Histidine mutation hisG46/R-factor mutation detecting base-pair substitutions
- Escherichia coli WP2uvrA: tryptophan-dependant mutation detecting base-pair substitutions
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 and ß-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Dose range-finding: TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. Based on the results of the dose range finding, concentrations to be tested in a first and second mutation experiment were selected.

First mutation experiment: Barium Nitrate was tested in triplicate both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98 at 100, 333, 1000, 3330 and 5000 μg/plate.

Second mutation experiment: Barium Nitrate was tested in triplicate both in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA at 100, 333, 1000, 3330 and 5000 μg/plate.
Vehicle / solvent:
Milli-Q water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
5 µg/plate; without metabolic activation; solvent: saline; TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
2.5 µg/plate; without metabolic activation; solvent: DMSO; TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
10 µg/plate; without metabolic activation; solvent: DMSO; TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
650 µg/plate; without metabolic activation; solvent: DMSO; TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 µg/plate; without metabolic activation; solvent: DMSO; WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1535 (2.5 µg/plate), TA98 (1 µg/plate), WP2uvrA (10 µg/plate); with metabolic activation at 5% and 10%; solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1537 (2.5 µg/plate) with metabolic activation at 5%; solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1537 (5 µg/plate) with metabolic activation at 10%; solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100 (1 µg/plate) with metabolic activation at 5%; solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100 (2.5 µg/plate) with metabolic activation at 10%; solvent DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (1E09 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q water and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h.

DURATION
Exposure duration: 48 ± 4 h.

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of barium nitrate, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined
Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Except TA1537 without S9-mix (first and second experiment) where an extreme reduction in the number of revertants was observed at 5000 μg/plate and with S9-mix (second experiment) where a moderate reduction was observed at 3330 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Dose range-finding:

- The dose range finding test was reported as a part of the first experiment of the mutation assay.

- Precipitation of barium nitrate on the plates was not observed at the start or at the end of the incubation period in both tester strains.

- No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

- No increase in the number of revertants was observed.

Deviations:

- The temperature was recorded to be outside the range of 37.0 ± 1.0°C specified in the protocol for approximately 1.5 hour in the second mutation experiment (with a maximum of 39.4°C).

Evaluation: This short term deviation was observed within three hours after initiation of the test and was caused by adjustment of the temperature in the incubator after placing an amount of selective agar plates in the incubator. The negative control data (number of spontaneous revertants per plate) were within the laboratory historical range for each tester strain, therefore this short deviation of the temperature had no effect on the results of the study. The study integrity was not adversely affected by the deviation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results of this study it is concluded that barium nitrate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.