Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 15, 2018
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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
of Commission Regulation (EC) number 440/2008 of 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries (November 24, 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli
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:
Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Pre-determined dose range for Experiment 1: 1.5 to 5000 μg/plate
Experiment 2 with amended dose range: Six test item concentrations per bacterial strain from 15 to 5000 µg/plate (based on Experiment 1)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (Fisher Scientific, Batch No 1714907; purity: >99 %; Expiry: October 2022)
- 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.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Test Item Preparation and Analysis

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. The test item was accurately weighed and, on the day of each experiment, approximate halflog dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction was required for purity. 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. 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.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described above except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method

As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 µg/plate. Six test item dose levels per bacterial strain were selected in the second mutation test in order
to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described above except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


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 (Cariello and Piegorsch, 1996)).

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.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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
Key result
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (light and powdery in appearance) was noted at 5000 µg/plate in Experiments 1 and 2 in both the absence and presence of S9-mix. These observations did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: In the first experiment (plate incorporation method), the maximum dose level of the test item was 5000 µg/plate (the maximum concentration). There was no visible reduction in the growth of the bacterial background lawns noted to any of the tester strains in either the absence and presence of S9-mix at any test item dose level.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See attached background material for Appendix 1: History Profile of Vehicle and Positive Control Values
- Combined vehicle and untreated historical control data: See attached background material for Appendix 1: History Profile of Vehicle and Positive Control Values

Any other information on results incl. tables

No biologically relevant growth in the frequency of mutation (i.e. revertant colonies) was observed for any of the bacterial strains with or without metabolic activation (S9-mix), at any test dose, under the plate incorporation method (Experiment 1) and pre-incubation method (Experiment 2). Small, statistically significant increases in TA100 revertant colony frequency were observed in the presence of S9-mix at 500, 1500, and 5000 µg/plate in the first mutation test (Experiment 1). However, these responses were within the in-house historical vehicle/untreated control values for the bacterial strain and were, therefore, disregarded.

Applicant's summary and conclusion

Conclusions:
Calcium titanium trioxide was determined to be non-mutagenic with and without metabolic activation under the conditions of the test.
Executive summary:

An in vitro gene mutation study in bacteria was undertaken to evaluate the potential genetic toxicity of calcium titanium trioxide. The experiment was conducted in line with Good Laboratory Practise (GLP), OECD Guideline 471 (Bacterial Reverse Mutation Assay), EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) of Commission Regulation (EC) number 440/2008 of 30 May 2008, EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998), and the Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries (November 24, 2000).

Two tests were performed using the Ames plate incorporation method (Experiment 1) and the pre-incubation method (Experiment 2) at up to eight dose levels with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors) (n= 3). Calcium titanium trioxide was applied at a predetermined dose range of 1.5 to 5000 µg/plate in Experiment 1 and in Experiment 2 the dose range was amended based on the outcome of Experiment 1 to 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 to achieve four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology. A negative (untreated) control and vehicle / solvent control that consisted of dimethyl sulphoxide were used. Five positive controls were also incorporated into the test design: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9-aminoacridine (9AA), 4-nitroquinoline-1-oxide (4NQO), 2-aminoanthracene (2AA), and benzo(a)pyrene (BP).

The sensitivity of the assay and the efficacy of the S9-mix were validated by the results of the vehicle and positive control chemicals. There were no toxicologically meaningful increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 under the plate incorporation method. Similarly, no 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 (S9-mix) in Experiment 2 under the pre-incubation method. Small, statistically significant increases in TA100 revertant colony frequency were observed in the presence of S9-mix at 500, 1500, and 5000 µg/plate in the first mutation test. However, these responses were within the in-house historical vehicle / untreated control values for the bacterial strain and were, therefore, considered of no biological relevance.

It has been concluded that calcium titanium trioxide was not mutagenic under the conditions of this experiment.