High temperature calcination products of titanium dioxide, calcium carbonate, calcium fluoride, quartz and antimony oxide containing lewisite and wollastonite

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2016 - 08 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or a gene segment, a block of genes or chromosomes (i.e. cytogenicity). Genetic toxicity is a broader term and refers to processes which alter the structure, information content or segregation of DNA and are not necessarily associated with mutagenicity. Column 1 of REACH Annex VII and VIII inform on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa and >10 tpa, respectively. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil both Annex VII and VIII information requirements.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Council Regulation (EC) No. 440/2008 method B.13/14: (published in the Official Journal of the European Union L 142, dated May 31, 2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EN ISO 10993-3: Biological evaluation of medical devices - Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity, current edition.

Version / remarks:

Current edition.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Extraction: EN ISO 10993-12: Biological evaluation of medical devices - Part 12: Sample preparation and reference materials

Version / remarks:

Current edition.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

antimony trioxide

IUPAC Name:

antimony trioxide

Constituent 2

Reference substance name:

Titanium dioxide

EC Number:

236-675-5

EC Name:

Titanium dioxide

Cas Number:

13463-67-7

Molecular formula:

O2Ti

IUPAC Name:

Titanium(IV) oxide

Constituent 3

Reference substance name:

Calcium oxide

EC Number:

215-138-9

EC Name:

Calcium oxide

Cas Number:

1305-78-8

Molecular formula:

CaO

IUPAC Name:

oxocalcium

Constituent 4

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# 434/08/15
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Antimony oxide (Sb2O3) 38.60%; Titanium oxide (TiO) 31.15%; Calcium oxide (CaO) 26.67%; Silicon oxide (SiO2): 4.40%; and Fluorine (F-): 1.53%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was not soluble in any of the solvents tested; highly purified water, Dimethylsulfoxide (DMSO), 0.05 Molar Hydrochloric acid (HCl), Ethanol or Acetone. Consequently, Uverithe was suspended in DMSO at concentrations of 100, 316, 1000, 3160 and 5000 µg/100 µL/plate.
- Final dilution of a dissolved solid, stock liquid or gel: 100, 316, 1000, 3160 and 5000 µg Uverithe.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspended fine powder

ELUATE TESTING
- Testing extract: An extract of the test item was prepared according to ISO 10993-12. The test item was eluted in 0.9% NaCl solution (Braun Melsungen batch No# 161948002) at a ratio of 0.2 g/mL.
- Elution procedure: The elution was conducted according to EN ISO 10993-3
- Method: The elution was performed for 24 hours at 70°C ± 1°C. After the end of elution, solid residues were removed by centrifugation at 1500 g and the supernatant was used for testing.
- Eluate concentration: The highest feasible volume (1000 μL) of the undiluted eluate/plate

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his-to his+ reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. S. typhimurium strains TA98 and TA1537 primarily respond to frameshift mutations at the histidine gene locus his D 3052 and his C 3076, respectively. Strains TA100, TA102 and TA1535 respond to base-pair substitutions in the his G 46, his G 428 and his G 46 locus.
In addition to the mutation in the histidine operon, these strains contain several other mutations that increase their ability to detect mutagens, such as rfa-, which refers to partial loss of lipopolysaccharide (LPS) barriers, increasing permeability to macromolecules. All strains presented a loss of DNA excision repair systems (ubr B-), excluding the TA102 wild-type. Plasmids (pKM 101, pKM 101/pAQ1) were also used to increase error-prone DNA repair and tetracycline resistance.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was obtained from Trinova Biochem (Lot No# 3613 and 3643)

Test concentrations with justification for top dose:

Uverithe was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations of the test item suspensions in DMSO ranging from 0.316 to 5000 μg/plate were tested. Test item precipitation was noted starting at a concentration of 100 μg/plate in both experiments. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. The recommended maximum test concentration for soluble non-cytotoxic test items is 5 mg/plate. For non-cytotoxic test items that are not soluble at 5 mg/plate, one or more concentrations tested should be insoluble in the final treatment mixture. Hence, 5000 μg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

TEST CULTURE
- Culture: S. typhimurium test strains were stored as lyophilizate pellets at 4°C and used to inoculate overnight cultures, grown in a water bath for 15 hours at 37°C in Oxoid (UNIPATH GmbH) at 10^8 - 10^9 cells/mL.
- Metabolic activation: S9 fraction (Trinova Biochem GmbH, Lot# 3613 and 3643).
- S9 preparation: The S9 mix was filter-sterilised by using a 0.45 µm filter and stored on dry ice.

METHOD OF APPLICATION:
The test item was examined in two independent experiments, each carried out with and without metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). Each experiment consisted of 3 plates/concentration and strains. The first experiment was carried out as the standard plate incorporation method, whereas the second was carried out as the preincubation method.

PLATE INCORPORATION METHOD
Sterile top agar containing 0.6% agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl/0.5 mM biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45°C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 10^8 viable cells in the late exponential or early stationary phase) 0.1 mL of test item suspensions in DMSO or 1 mL eluate in 0.9% NaCl solution (or 0.1 mL solvent or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer mentioned above.

The test components were mixed by vortexing the soft agar for 3 sec at low speed and then poured onto a coded 27.5 mL minimal glucose agar plate (Minimal Glucose Agar medium E). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed on a level surface with the cover on and finally allowed to harden. Immediately, the plates were inverted and placed in a dark 37°C incubator for 48 to 72 hours. The revertant colonies on the test plates and on the control plates were counted with a colony counter, and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared with the lawn on the negative control plate confirmed bacterial toxicity. Routine examination of the background lawn of bacterial growth resulting from the trace of histidine added to the top agar can be an aid in determining the presence of toxic effects. If massive cell death has occurred, the background lawn on the test plates will be sparse compared with control plates. In this case more histidine is available to the individual surviving bacteria and they undergo more cell divisions, consequently appearing as small colonies which can be mistaken for revertants if the absence of a normal background lawn is not noted.

PRE-INCUBATION METHOD
The test item was preincubated with the test strain (containing approximately 10^8 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of test item suspensions in DMSO or 1 mL eluate in 0.9% NaCl solution (or 0.1 mL solvent or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

EVALUATION
Bacteria colonies were counted employing the Biosys Biocount 5000 system. Print outs of the colony counts were filed with the raw data.

Rationale for test conditions:

Test conditions were consistent with OECD TG 471.

Evaluation criteria:

MUTAGENICITY CRITERIA
A test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
- positive results were reproducible and the histidine independence of the revertants was confirmed by streaking random samples on histidine-free agar plates. Biological relevance of the results should be considered.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

CYTOTOXICITY CRITERIA
- Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Statistics:

U-test according to MANN and WHITNEY and Spearman’s rank correlation coefficient.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
Test item precipitation was noted in the plate incorporation and pre-incubation tests in the presence or absence of S9 metabolic activation, at all tested concentrations of 100 - 5000 µg Uverithe in 100 µL DMSO per plate. No signs of cytoxicity were noted for the suspension or eluate of Uverithe in 0.9% NaCl, in any test strain.

MUTAGENICITY
No statistically significant increase in revertant colony numbers relative to control counts was reported for the suspension or eluate of Uverithe (≤ 5000 µg/plate) in any of the five test strains, in two independent experiments, in the presence or absence of S9 metabolic activation. The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. Column 1 of REACH Annex VII and VIII inform on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa and >10 tpa, respectively. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil both Annex VII and VIII information requirements. Under the test conditions, Uverithe did not present any cytotoxic or mutagenic properties in the Bacterial Reverse Mutation Test. Conducted according to the aforementioned guidelines and GLP, the Ames Test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. The Bacterial Reverse Mutation Test detects gain of function point mutations and frameshifts in vitro. A trace of histidine allows the logarithmic division of the histidine-requiring bacteria in the presence of the test item. This period of auxotrophic cell division forms a lawn of histidine-requiring bacteria, whose growth is prevented by exhaustion of histidine. Only the small fraction of bacteria which has reverted to histidine- independence (either spontaneously or by the action of the test chemical) will continue to divide to form discrete, randomly distributed visible colonies, each one of which consists of the progeny of a single mutant bacterium. The assay determines whether the addition of graded concentrations of the test item to a series of such plates induces a concentration-related increase in mutant colonies compared with plates treated only with the appropriate solvent.

Uverithe (100, 316, 1000, 3160 or 5000 µg/plate) was examined in five Salmonella typhimurium strains, TA98, TA100, TA102, TA1535 and TA1537, in plate incorporation and pre-incubation experiments conducted in the presence and absence of S9 metabolic activation. An extract of the test item, prepared in 0.9% NaCl according to ISO 10993-12, was also tested in the in vitro system. No signs of cytoxicity were noted for Uverithe or the eluate. No mutagenic effect was identified in the S. typhimurium strains in two individual experiments carried out with and without S9 metabolic activation. The positive control items showed the expected increases in revertant colonies and confirmed the validity of the test.

The Ames Test (OECD 471, EU B.13/14) is an intentionally accepted in vitro test method to detect mutagenicity, as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the Ames Test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).