Registration Dossier

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:
7th September 2017- 30th October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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)
Qualifier:
according to
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Batch No 20170425
- Expiration date of the lot/batch: 24th April 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

Method

Target gene:
Histidine
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction prepared according to Ames et al.
Test concentrations with justification for top dose:
The test item concentrations were chosen based on the basis of a pre-experiment performed at the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
Three plates per dose were prepared using the plate incorporation method.

Main Test – Experiment 1 (plate incorporation test)
10.0, 31.6, 100, 316, 1000 and 2500 µg/plate were assayed with and without metabolic activation.

Main Test – Experiment 2 (pre-incubation test)
3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate were assayed with and without metabolic activation.
Vehicle:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: THe vehicle was compatable with the survival of the bacteria, tthe S9 activity and the solubility of the inorganic test item.
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
purified water
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD)and 2-aminoanthracene (2-AA)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1) and pre-incubation (Experiment 2)
DURATION
- Pre-incubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C in the dark for both application methods.
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells):N/A

NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
The number of revertant colonies and effects on the growth of the bacterial background lawn were used to assess the toxicity of the test substance to the test system.
Evaluation criteria:
Mutation Factor is calculated by dividing the mean of the revertant counts by the mean values of the solvent control (exact values).
A test item is considered mutagenic if:
- A clear and dose-related increase in the number of revertant occurs
and /or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as:
– an increase in the number of reversions at least twice as high as the solvent control in tester strains TA 98, TA 100 and TA 102
and/or
– an increase in the number of reversions at least three times as high as the solvent control in tester strains TA 1535 and TA 1537.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other:
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Expt 2: 2500 - metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not determined
- Effects of osmolality: Not determined
- Evaporation from medium: Not determined
- Precipitation: None detected

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed on 8 concentrations in the range 3.16 - 5000µg/plate using bacterial strains TA98 and TA 100.



Any other information on results incl. tables

Results for concurrent negative controls and Experiments 1 and 2 with and without metabolic activation are attached as Tables 1 - 3 below.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, barium selenate did not cause gene mutations by base pair changes or frameshfts in the genome of the tester strains used.
Barium selenate is not considered mutagenic in the bacterial reverse mutation assay.
Executive summary:

 The genetic toxicity of barium selenate was examined in a bacterial reverse mutation assay (Ames test) performed to GLP and in accordance with OECD 471 and EU method B.13/14. Salmonella Typhimurium strains TA 98, TA 100, TA1535, TA 1537 and TA 102 were tested with and without S9 rat liver fraction metabolic activation.

A preliminary toxicity test in the range 3.16 µg/plate to 5000 µg/plate showed toxic effects in the TA 100 strain at the highest dose level, therefore the main study was performed with a highest dose level of 2500 µg/plate. Two mutation tests were performed, Experiment 1 used the plate incorporation method and Experiment 2 was performed using the pre-incubation method, test substance concentrations of 10, 31.6, 100, 316, 1000 and 2500 µg/plate were assessed in both experiments. Positive and vehicle controls were also tested.

Precipitation of the test item was observed in all tester strains used in Experiment 1 and 2 (with and without metabolic activation) at 2500 µg/plate and in experiment 2 at 1000 µg/plate and above.

In Experiment 1, no toxic effects were noted in tester strains TA 98, TA 100, TA 1537 and TA 102 (with or without metabolic activation) in either experiment or at any dose concentration. Toxic effects were observed in strain TA 1535 at 2000 µg/plate (with metabolic activation). Experiment 1 also showed a reduction in the number of revertants down to a mutation factor of ≤0.5 in strain TA 1535 at 10, 31.6 and 100 µg/plate  and in TA 1537 at 1000 µg/plate (without metabolic activation) however these were not considered biologically significant due to a lack of dose response.

In Experiment 2 toxic effects were observed in strain TA 100 at 2500 µg/plate (without metabolic activation) and TA 2535 and TA 1537 at 2500 µg/plate (with metabolic activation).

No biologically relevant increases in revertant colony numbers were observed in any of the tester strains after treatment with barium selenate, either in the presence of or without metabolic activation.

Under the conditions of the test, barium selenate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Barium selenate is not considered mutagenic in the bacterial reverse mutation assay.