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Genetic toxicity: in vitro

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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:
20 June 2012 and 20 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficincies, which do not affect the quality of relevant results.

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Strontium titanium trioxide
EC Number:
235-044-1
EC Name:
Strontium titanium trioxide
Cas Number:
12060-59-2
Molecular formula:
O3Ti.Sr
IUPAC Name:
strontium titanium trioxide
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Strontium Titanium Trioxide
- Physical state: Pale grey solid
- Analytical purity: equal to or greater than 99 %
- Lot/batch No.: 24924
- Expiration date of the lot/batch: 30 March 2013
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity
to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel (1976)).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
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. (with and without S9-mix)

Mutation Test - Experiment 1
50, 150, 500, 1500, 5000 µg/plate (in triplicate) (with and without S9-mix)

Mutation Test - Experiment 2
50, 150, 500, 1500, 5000 µg/plate (in triplicate) (with and without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/ml, acetone at 100 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
WP2uvrA - Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
3 µg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA100 - Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
5 µg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA1535 - Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
80 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
TA1537 - Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
0.2 µg/plate
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
TA98 - Without S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
1 µg/plate
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
TA100 - With S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
TA1535 and TA1537 - With S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
WP2uvrA - With S9-mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
5 µg/plate
Positive control substance:
benzo(a)pyrene
Remarks:
TA98 - With S9-mix
Details on test system and experimental conditions:
Tester Strains
The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1078364 08/16) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Preparation of Test and Reference Items
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 15 minutes at 40°C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

Microsomal Enzyme Fraction
The S9 Microsomal fraction was prepared in-house (15 April 2012) from rats induced with Phenobarbitone/p-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCI solution (1 g liver to 3 ml KCI) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196°C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

S9-Mix and Agar
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.

S9: 5.0mL
1.65 M KCI/0.4 M MgCI2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6 % Bacto agar (lot numbers 1171985 04/16 and 1297425 09/16) and 0.50/0 sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS Ltd (lot numbers 1177487 06/16, 1195759 11/16 and 1215904 04/16).

Test Procedure
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. 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 (dimethyl sulphoxide) 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 a colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1
Five concentrations of the test item (50, 150, 500, 1500, 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the vehicle, test item formulation or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter.

Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was the same as Experiment 1 (50 to 5000 µg/plate).
As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described in Experiment 1.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter.

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
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 (De Serres and Shelby (1979)).
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 judgement 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:
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:
Preliminary Toxicity Test
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay can be found in "any other information on results" below.

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid
supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (please see "any other information on results" below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive controls and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2 (please see Attachment 1 - Results Tables).
A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Appendix 1 (please see attached).
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 (µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 50 (µg/plate in Experiment 1. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 (µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.
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'.

Any other information on results incl. tables

Toxicity Assay Results Table

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

95

109

124

114

107

115

118

119

112

118

123

+

TA100

109

110

117

108

102

91

82

117

106

111

112

-

WP2uvrA

23

19

16

32

34

30

24

26

20

23

25

+

WP2uvrA

32

33

30

33

34

30

24

36

31

35

34

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

84

24

29

24

12

96 (97)

27 (22)

33 (34)

29 (26)

13 (12)

112

16

41

24

12

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100*

TA1535

WP2uvrA

TA98

TA1537

74

15

35

28

8

86 (78)

21 (17)

36 (30)

23 (25)

8 (11)

74

15

20

23

17

* Experimental procedure performed at a later date (with and without S9-mix) due to poor culture growth in the original test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item, Strontium Titanium Trioxide, was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Strontium Titanium Trioxide, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 to 5000 (µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 (µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with' or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 50 (µg/plate in Experiment 1. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Conclusion.

The test item, Strontium Titanium Trioxide, was considered to be non-mutagenic under the conditions of this test.

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