Reaction Mass of Dialuminium Titanium Pentoxide and Strontium Calcium Aluminosilicate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Mammalian cell mutation: Wollny, 2012

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30th April 2012 to 27th November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dialuminium titanium pentaoxide

EC Number:

234-456-9

EC Name:

Dialuminium titanium pentaoxide

Cas Number:

12004-39-6

Molecular formula:

Al2O5Ti

IUPAC Name:

Dialuminum titanium pentaoxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Aluminium titanium oxide.
- Molecular formula (if other than submission substance): Al2TiO5
- Molecular weight (if other than submission substance): 181.83
- Physical state: White powder

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency.
Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 5x10E5 cells are seeded into each flask with 15 mL of MEM-Hanks’ (minimal essential medium containing 25mM HEPES) supplemented with 10 % foetal bovine serum (FBS) and 1 % neomycin. The cells are sub-cultured twice weekly. The cell cultures are incubated at 37 °C in a water saturated atmosphere containing 1.5 % carbon dioxide (98.5% air).
For the selection of mutant cells the medium is supplemented with 11 µg/mL 6-thioguanine (6-TG).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 without S9 mix: 57.8, 115.6, 231.3, 462.5, 925.0 and 1850µg/mL
Experiment 1 with S9 mix: 57.8, 115.6, 231.3, 462.5, 925.0 and 1850µg/mL
In experiment I with and without S9 mix the cultures at the lowest concentration of 57.8 µg/mL were not continued as a minimum of only four analysable concentrations is required.

Experiment 2 without S9 mix: 57.8, 115.6, 231.3, 462.5, 925.0 and 1850µg/mL
Experiment 2 with S9 mix: 57.8, 115.6, 231.3, 462.5, 925.0 and 1850µg/mL

Vehicle / solvent:

Sodium carboxymethyl cellulose

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

1% aqueous solution of sodium carboxymethyl cellulose

True negative controls:

no

Positive controls:

yes

Remarks:

0.15mg/mL

Positive control substance:

ethylmethanesulphonate

Remarks:

Used in the absence of metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

1.1 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Used in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (MEM containing Hank’s salts, Neomycin (5 µg/mL) and Amphotericin B (1 %)). All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

The stock cultures were trypsinized at 37 °C for 5 minutes two days after sub-cultivation for experiment 1 and four days after sub-cultivation for experiment 2. The enzymatic digestion was then stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks. Approximately 1.5x10E6 and 5x10E2 cells were seeded in each flask respectively. The medium was MEM-Hanks with 10 % FBS (complete medium). The cells were grown for 24 hours prior to treatment.

After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment without metabolic activation the cells were exposed to the test item for 24 h in complete medium supplemented with 10 % FBS. Concurrent solvent and positive controls were treated in parallel.

Four days (experiment I) and three days (experiment II) after treatment 1.5x10E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days ten 80 cm² cell culture flasks were seeded with about 3 - 5x10E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

NUMBER OF REPLICATIONS: The study was conducted in duplicate.

DETERMINATION OF CYTOTOXICITY
The colonies used to determine the cloning efficiency I (survival) were fixed and stained approx. 7 days after treatment.
Relative cloning efficiency I (survival) was (mean number of colonies per flask divided by the mean number of colonies per flask of the corresponding solvent control) x 100.

Evaluation criteria:

A response was classified as positive if it induced a concentration-related increase of the mutant frequency three times higher than the spontaneous mutation frequency in the experiment and that exceeds the spontaneous mutation rate in the range normally found (2.6 – 43.5 mutants per 10xE6 cells).
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.

The test item is classified as mutagenic if it reproduces positive responses.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent and/or negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (2.6 – 45.3 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent and/or negative controls within all experiments of this study is also taken into consideration.
Treatments that reduce relative survival to less than ten percent may not be used as sufficient evidence for mutagenicity as it relates to risk assessment.
If replicates are not similar, it will also be taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent con-trol groups. A trend was judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

The test item formed a stable suspension in 1% CMC-Na aqueous solution and in the medium during 4 hour treatment. Precipitation of the test item visible to the unaided eye at the end of treatment did not occur in the first experiment up to the maximum concentration with and without metabolic activation. Microscopic evaluation proved that the test item formed a suspension rather than a solution as particles were visible down to the lowest concentration.
In the second experiment precipitation at the end of treatment was noted at the two highest concentrations of 925 and 1850 µg/mL following 24 hour treatment without metabolic activation. Again, microscopic evaluation proved that the test item formed a suspension down to the lowest concentration, so, during 24 hour treatment without metabolic activation the suspension was not stable over the entire exposure period at high concentrations as already indicated by the pre-experiment.
No relevant cytotoxic effects as indicated by a relative cloning efficiency I and/or relative cloning efficiency II of less than 50% in both parallel cultures were noted up to the maximum concentration with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/10xE6 cells was observed in the main experiments up to the maximum concentration of 10 mM.
The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment I with metabolic activation at 231.3 µg/mL. This effect however, was judged as biologically irrelevant since it was not reproduced in the parallel culture performed under identical experimental conditions and the increase was not dose dependent as indicated by the lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in experiment I and II.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.0 up to 29.5 mutants per 10E6 cells; the range of the negative controls was 7.2 up to 27.8. The range of the groups treated with the test item was from 4.1 up to 50.4 mutants per 10E6 cells .

Any other information on results incl. tables

Mutant colonies/10^6cells Experiment 1 Experiment 2 4h treatment 24h treatment 4h treatment -S9 +S9 -S9 +S9 Negative control 17.1 21.1 18.2 27..8 9.8 12.7 20.2 7.2 Solvent control 18.5 23.7 8.0 17.6 20.9 12.1 29.5 23.2 57.8 µg/mL - - - - - - - - 115.6 µg/mL 13.9 17.3 18.4 16.5 14.6 13.1 30.5 13.4 231.3 µg/mL 11.5 42.4 39.5 29.2 17.3 13.8 18.8 15.2 462.5 µg/mL 10.8 29.5 4.1 25.9 16.8 11.3 50.4 15.0 925 µg/mL 37.9 45.3 12.3 27.4 20.9p 12.5p 28.6 20.0 1850 µg/mL 21.5 25.3 9.3 31.5 16.8p 14.4p 42.9 23.0 Positive control 131.0 240.2 1106.6 1682.4 341.8 275.8 311.3 216.0 p: precipitation of the test material

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material, Aluminium Titanium Oxide, was not considered to have mutagenic potential.

Executive summary:

The study was conducted in accordance with GLP, OECD Guideline 476 and EU Method B17 to determine the potential of Aluminium Titanium Oxide to cause mutagenicity when tested in V79 cells of the Chinese hamster. Two experiments were conducted, using two parallel cultures. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The testing concentrations used in both tests, with and without metabolic activation were 57.8, 115.6, 231.3, 462.5, 925.0 and 1850µg/ml. The maximum concentration of 1850 µg/mL was equal to a molar concentration of approximately 10 mM. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system. Under the conditions of this study, the test material, Aluminium Titanium Oxide, was not considered to have mutagenic potential in this HPRT assay.