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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD473 in vitro mammalian cytogenicity/chromosome aberration study with the read-across substance, antimony nickel titanium oxide yellow, using Chinese hamster lung (CHL/IU) cells. The test item was formulated in Distilled water and it was examined up to the cytotoxic concentrations according to the relevant OECD guideline covering the range from cytotoxicity to no or little cytotoxicity. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (vehicle) and positive control sample if possible. No further detals of study methods were available, however, the performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured Chinese hamster lung cells used in this study. In this study higher concentrations were associated with frank cytotoxicity (low survival of cells in media) but additionally at some concentrations below this, there was no or low mitotic activity. Only concentrations below these effects could be scored for mitotic damage.

OECD476 in vitro mammalian gene mutation study (mutation at the thymidine kinase locus) with the read-across substance, antimony nickel titanium oxide yellow, using mouse lymphoma L5178Y cells. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). A 4-hour treatment incubation period was used for all experiments. In the cytotoxicity Range-Finder Experiment, concentrations were tested in the absence and presence of S9, ranging from 0.195 to 100 mg/L (limited by solubility in the primary vehicle, DMSO). In the two independent trials 3 vehicle controls and 2 positive controls (methylmethanesulfonate) were employed with an exposure period of 4 hours, an expression period of 2 days and a selection period of 10-14 days. Experiment I was not considered acceptable because of cell culture problems although no mutagenicity of the test substance was observed. In Experiment II, 6 concentrations, ranging from 3.13 to 100 µg/mL, were tested in the absence and presence of S9. Although no cytotoxicity was observed, the concentrations used showed precipitation.

OECD 471 in vitro bacterial reverse mutation test with Uverithe. The test item 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, was also tested in the in vitro system. No signs of cytotoxicity were noted for the test item or the eluate, but the test substance was tested up to precipitating concentrations in DMSO. No mutagenic effect was identified in the S. typhimurium strains in the experiments with and without S9 metabolic activation. The experiments were considered valid as the positive control showed the expected increases in revertant colonies.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

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

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

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

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

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 / 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)

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.

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

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

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

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

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.

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).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to GLP guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Clive et al., 1987, Mutation Research, 189, 143-156

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium: RPMI 1640 supplemented with PluronicF68, L-glutamine, sodium pyruvate, antibiotics and 10% horse serum; treatment medium: Fischer's medium with same supplements but 5% horse serum; cloning medium: same culture medium but with 20% horse serum and without PluronicF68 and addition of BBL purified agar (0.24%); selection medium: cloning medium containing 3 µg/ml of TFT (5-trifluorothymidine)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

3.13, 6.25, 12.5, 25, 50, 100 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 h
- Selection time (if incubation with a selection agent): 10-14 h


SELECTION AGENT: 5-trifluorothymidine (TIFT)

NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: approx. 3*10e5

Evaluation criteria:

- The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is >= 2 times the concurrent background mutant frequency (as the average of the vehicle control cultures).
- A dose-related or toxicity-related increase in mutant frequency should be observed.
- If the mutant frequency obtained for a single dose at or near the highest testable toxicity is about two or more times the minimum criterion, the test article will be considered mutagenic in a single trial.
- Treatments that induce less than ten percent relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Trial 1 was not considered acceptable because of cell culture problems (slower growth; positive control cultures were excessively cytotoxic; the vehicle control mutant frequencies, while still in the acceptable range, were higher than usual. However, no mutagenicity of the TS was observed.

Trial 2
Treatment (µg/ml)	Metabolic activation	Suspension growth*a	Cloning efficiency*b	Relative growth*c	Mutant frequency (10e-6 units)
3.13	no	109	85	93	92
	yes	110	80	88	108
6.25	no	102	77	79	115
	yes	105	76	80	81
12.5	no	104	75	78	111
	yes	105	89	93	84
25	no	86	79	68	10 5
	yes	86	92	79	91
50	no	92	87	79	97
	yes	89	85	76	110
100	no	82	73	60	100
	yes	67	90	61	123
*a = relative to vehicle control
*b = relative to vehicle control, total viable colony count/ number of cells seeded * 100
*c = (relative suspension growth + relative cloning efficiency) / 100
*d = relative to vehicle control,  (total mutant colonies/ total viable colonies)*2*10e-4; dezimal is moved to express the frequency in units of 10e-6

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to GLP guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under ‘Attached justification’ in Section 13 of IUCLID and under ‘Cross-reference’ in the IUCLID Robust Study Summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Clive et al., 1987, Mutation Research, 189, 143-156

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium: RPMI 1640 supplemented with PluronicF68, L-glutamine, sodium pyruvate, antibiotics and 10% horse serum; treatment medium: Fischer's medium with same supplements but 5% horse serum; cloning medium: same culture medium but with 20% horse serum and without PluronicF68 and addition of BBL purified agar (0.24%); selection medium: cloning medium containing 3 µg/ml of TFT (5-trifluorothymidine)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

3.13, 6.25, 12.5, 25, 50, 100 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 h
- Selection time (if incubation with a selection agent): 10-14 h


SELECTION AGENT: 5-trifluorothymidine (TIFT)

NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: approx. 3*10e5

Evaluation criteria:

- The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is >= 2 times the concurrent background mutant frequency (as the average of the vehicle control cultures).
- A dose-related or toxicity-related increase in mutant frequency should be observed.
- If the mutant frequency obtained for a single dose at or near the highest testable toxicity is about two or more times the minimum criterion, the test article will be considered mutagenic in a single trial.
- Treatments that induce less than ten percent relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Trial 1 was not considered acceptable because of cell culture problems (slower growth; positive control cultures were excessively cytotoxic; the vehicle control mutant frequencies, while still in the acceptable range, were higher than usual. However, no mutagenicity of the TS was observed.

Trial 2
Treatment (µg/ml)	Metabolic activation	Suspension growth*a	Cloning efficiency*b	Relative growth*c	Mutant frequency (10e-6 units)
3.13	no	109	85	93	92
	yes	110	80	88	108
6.25	no	102	77	79	115
	yes	105	76	80	81
12.5	no	104	75	78	111
	yes	105	89	93	84
25	no	86	79	68	10 5
	yes	86	92	79	91
50	no	92	87	79	97
	yes	89	85	76	110
100	no	82	73	60	100
	yes	67	90	61	123
*a = relative to vehicle control
*b = relative to vehicle control, total viable colony count/ number of cells seeded * 100
*c = (relative suspension growth + relative cloning efficiency) / 100
*d = relative to vehicle control,  (total mutant colonies/ total viable colonies)*2*10e-4; dezimal is moved to express the frequency in units of 10e-6

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed data base (Peer review was conducted by a Japanese toxicological expert group at March 22, 2001. However, only secondary source material available)

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

short-term treatment (-S9 mix): 4.88, 9.77, 19.5, 39.1, 78.1 µg/ml;
short-term treatment (+S9 mix): 19.5, 39.1, 78.1, 156, 313, 625, 1250 µg/ml;
continuous treatment (-S9 mix): 4.88, 9.77, 19.5, 39.1, 78.1 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9: Mitomycin C; +S9: Cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF CELLS EVALUATED: 200

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at more than 39.1, 78.1, and 19.5 µg/ml in -S9mix, +S9mix and continuous treatments, respectively

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed data base (Peer review was conducted by a Japanese toxicological expert group at March 22, 2001. However, only secondary source material available)

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under ‘Attached justification’ in Section 13 of IUCLID and under ‘Cross-reference’ in the IUCLID Robust Study Summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

short-term treatment (-S9 mix): 4.88, 9.77, 19.5, 39.1, 78.1 µg/ml;
short-term treatment (+S9 mix): 19.5, 39.1, 78.1, 156, 313, 625, 1250 µg/ml;
continuous treatment (-S9 mix): 4.88, 9.77, 19.5, 39.1, 78.1 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9: Mitomycin C; +S9: Cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF CELLS EVALUATED: 200

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at more than 39.1, 78.1, and 19.5 µg/ml in -S9mix, +S9mix and continuous treatments, respectively

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The read-across substance, antimony nickel titanium oxide yellow, was tested for potential clastogenic activity using the Chromosome Aberration Assay. Treatment with the test substance did not result in a statistically significant increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system. Therefore, the read-across substance, antimony nickel titanium oxide yellow, did not induce a repeatable, significant level of chromosome aberrations in the performed experiments and is considered non-clastogenic in this system.

The read-across substance, antimony nickel titanium oxide yellow was also assayed for the ability to induce mutation at the thymidine kinase (TK) locus in mouse lymphoma cells with a protocol including a cytotoxicity range-finding experiment. Antimony nickel titanium oxide yellow did not induce mutation at the TK locus of L5178 mouse lymphoma cells up to the maximum practical concentration in two independent experiments and in the presence and absence of a metabolic activation system.

In addition, studies to the genetox program included an in vitro reverse gene mutation study in bacteria (Ames test) with the target substance, Uverithe. This assay also proved negative under the conditions of the test.

Justification for classification or non-classification

Using a weight of evidence approach assessing the required in vitro mutagenicity tests (in vitro mammalian chromosome aberration test (OECD473) and in vitro mammalian cell gene mutation test (OECD476) in source substances, and a bacterial reverse mutation test (OECD471) with the target substance), the negative results in all tests would indicate that, according to the Globally Harmonised Classification System (GHS) and EU CLP criteria, the test item does not require classification for a germ cell mutagen.