potassium titanium oxide (K2Ti6O13)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of the test material was investigated in five in vitro studies (Ames test, chromosome aberration test and mammalian cell gene mutation assay) which were conducted under GLP conditions. Results from these in vitro genotoxicity studies were all negative with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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:

07 January 1999 to 01 February 1999

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 deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Annex V (Ames test).

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 23 March 1998 Date of Signature: 21 July 1998)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon (his) for Salmonella.
Tryptophan operon (trp) for E.Coli.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

other: Including a deletion through the excision repair gene (uvrB-) which renders the capability of DNA exision repair and deep rough mutation (rfa)

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

other: Including a deletion through the excision repair gene (uvrA-)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced, rat-liver S9.

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
Main test, experiments 1&2: 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

Sterile distilled water

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 5000 µg/plate

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h.

NUMBER OF REPLICATIONS: Triplicate.

DETERMINATION OF CYTOTOXICITY: Plates were assessed for effects on the growth of the bacterial background lawn.

OTHER EXAMINATIONS
- Other:
Solubility: Test material precipitation was examined on the plates.
Sterlility: (Preliminary study only) The aliquot of 0.1 ml of the maximum concentration of the test material (5000 µg/plate) and 2 ml of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plates in order to assess the sterility of the test material.

Evaluation criteria:

A test material may be considered positive in the test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically significant increase in the relevant count in at least one strain of bacteria.

Statistics:

Dunnett’s method of linear regression.

Species / strain:

other: S. typhimurium TA 100, E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

not examined

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

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:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without
metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A black particulate precipitate was observed at 5000 pg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used

COMPARISON WITH HISTORICAL CONTROL DATA: All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges. No statistically significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the substance, either with or without metabolic activation.
Solvent control plates gave counts of revertant colonies within the normal range.

INFORMATION ON CYTOTOXICITY: No toxicity was exhibited to any of the strains of bacteria used.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Negative with and without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The test was conducted at a GLP facility in accordance with adopted test guidelines. The study outcomes were presented in well-documented report format. Therefore a reliability of 1 is assigned.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 December 1987 to 18 March 1988

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 deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: Japan: Partial Amendment of the Information on Test Methods for New Chemical Substances

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan: Information on the Testing ,Facilities Stipulated in Article 3 of the Order Prescribing the Test Item Related to New Chemical Substances

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not applicable

Principles of method if other than guideline:

OECD TG 473 was not available at the time of the test. The test followed the similar methodologies in accordance with the following references;

Matsuoka, A. et a1.: Chromosomal aberration test on 29 chemicals combined with S9
mix in vitro. Mutation Res., 66: 277-290, 1979.

Ishidate, M. (ed.): Data Book for Chromosomal Aberration Tests, (Realize Printing Co., 1983).

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Thymidine kinase operon (tk).

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential medium.

A cell suspension, which was stored in liquid nitrogen after the addition of DMSO at a concentration of 10%, was seeded in culture medium, and cells with a passage number of 4 or less, as counted from the time point of the seeding, were used in the present test.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/5,6-benzoflavone induced, rat-liver S9.

Test concentrations with justification for top dose:

(Preliminary test)
0.25, 0.5, 0.7 and 1.0 mg/ml without S9 mix
0.75, 1.5, 2.0 and 3.0 mg/ml with S9 mix

(Main test)
Direct method (without S9 mix) 0.25, 0.5 and 1.0 mg/ml.
(Metabolic Activation method - with or without S9 mix) 0.75, 1.5 and 3.0 mg/ml.

Untreated negative controls:

yes

Remarks:

(concurrent vehicle control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine, 0.0025 mg/ml

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

(concurrent vehicle control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Migrated to IUCLID6: 0.01 mg/ml

Species / strain:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1 mg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

mammalian cell line, other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: other: Preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance potassium titanate fibre did not induce chromosomal aberrations in CHL cells under the conditions of this test. The test substance was therefore considered to be non-clastogenic to Chinese hamster lung cell (CHL/IU) in vitro.

Executive summary:

The substance was tested in vitro for chromosomal aberrations in CHL/IU cells derived from the Chinese hamster lung. No apparent increase in chromosomal aberrations was noted for the direct method at an exposure concentration of 0.25, 0.5, or 1.0 mg/ml or the metabolic activation method at 0.75, 1.5, or 3.0 mg/ml.

These results indicate that, under the conditions of this study, the substance did not induce chromosomal aberrations in CHL cells.

The test was conducted at a GLP facility in accordance with adopted test guidelines and a reliability of 1 is assigned.

Reference 3

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:

19 March to 08 July 1999

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 deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 23 March 1998 Date of Signature: 21 July 1998)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Thymidine kinase operon (tk).

Species / strain / cell type:

other: human lymphocyte cells from healthy volunteers

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium (supplemented with sodium bicarbonate, HEPE5 buffer, L-glutamine, penicillin/streptomycin, amphotericin Band 15% foetal calf serum)

Additional strain / cell type characteristics:

other: AGT: approximately 14 hours

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/ß-naphthoflavone induced, rat-liver S9.

Test concentrations with justification for top dose:

Chromosome Aberration Test Experiment 1:
(with and without S9 mix) 39.06, 78.1, 156.25, 312.5, 625, 1250, 2500, 5000 ug/ml.

Chromosome Aberration Test Experiment 2:
(with and without S9 mix) 19.5, 39, 78.1, 156.25, 312.5 and 625 ug/ml.

Vehicle / solvent:

Minimum Essential Media

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

other: (concurrent untreated control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours (Experiment 1); 4 hours with S9, 20 hours without S9 (Experiment 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

STAIN (for cytogenetic assays): 5% Gurrs Giemsa.

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000 cells for each experiment.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index.

OTHER: Chromosome damage. Where possible the first 100 consecutive well-spread metaphases from each culture were counted.
Frequency of polyploid cells were also assessed.

Evaluation criteria:

Cells with 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test and chi-square test.

Species / strain:

other: human lymphocyte cells from healthy volunteers

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 625 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Observations:
All the negative control cultures gave values of chromosome aberrations with the expected range for normal human lymphocytes.

All the positive control cultures gave highly-significant increases in the frequency of aberrations, indicating the satisfactory performance of the test and of the activity of the metabolising system.

The substance was seen to induce no significant, dose-related increases in the frequency of aberrations in any of the treatment groups, either with or without S9.

The substance did not induce a significant increase in the numbers of hyperdiploid cells at any dose level in any of the treatment cases.

Precipitations:
(Experiment 1) The precipitate was heavy at and above 625 pg/mI with or without S9.

Remarks on result:

other: other: Experiment 1

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. The substance was therefore considered to be non-clastogenic to human Iymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations. 

 

Duplicate cultures of human Iymphocytes, treated with the test material (TXAX-A), were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 (at a 2% final concentration) was repeated, whilst in the absence of activation the exposure time was increased to 20 hours.

 

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

 

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human Iymphocytes.

 

All the positive control treatments gave statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material, TXAX-A, did not induce any statistically significant increases in the frequency of cells with aberrations in either of two separate experiments. The test material was shown to be non-c1astogenic to human Iymphocytes in vitro.

The test was conducted at a GLP facility in accordance with adopted test guidelines. The study outcomes were presented in well-documented report format. Therefore a reliability of 1 is assigned.

Reference 4

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

Study period:

16 August 2010 to 11 October 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 15 September 2009 Date of Signature: 26 November 2009)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used in the mutagenicity test was the maximum recommended dose level of 5000 µg/ml for the 4-hour exposure groups in both the absence and presence of metabolic activation, and limited by test material induced toxicity for the 24-hour exposure group in the absence of metabolic activation.

Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0277203 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle used:
Vehicle (R0 medium) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a suspension suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the United Kingdom Environmental Mutagen Society. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 19.53 to 5000 µg/ml. In all three of the exposure groups there were dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls, with the most marked reductions observed in the 24-hour exposure group. A precipitate of the test material was observed at all of the test material dose levels and increased in intensity with increase in dose concentration in all three of the exposure groups. With adequate evidence of exposure of the test material to the cells, as indicated by the %RSG values, in the subsequent mutagenicity test the maximum dose was the maximum recommended dose level of 5000 µg/ml for the 4-hour exposure groups in both the absence and presence of metabolic activation, and limited by test material induced toxicity for the 24-hour exposure group in the absence of metabolic activation.

With toxicity occurring beyond the onset of precipitation this became the limiting factor in dose selection for the mutagenicity experiments. This follows the recommendations in the OECD 476 test guideline.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

As was seen previously, there was evidence of modest toxicity following exposure to the test material in the presence of metabolic activation, as indicated by the %RSG values (Table 6). The levels of toxicity observed in the presence of metabolic activation were very similar to those of the preliminary toxicity test. However, the levels of toxicity observed in the preliminary toxicity test in the absence of metabolic activation were not reproduced (Table 3). It was considered that this may have been due to the presence of precipitate resulting in variable exposure of the test material to the cells or just due to the normal variations seen in biological systems. However, as the maximum recommended dose level of 5000 µg/ml had been investigated, and adequate evidence of toxicity had been achieved in the presence of metabolic activation, the test material was considered to have been adequately tested. There was no evidence of any reductions in viabilities (%V), therefore indicating that residual toxicity had not occurred in either the absence or presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). Precipitate of test material was observed at all of the test material dose levels.

The numbers of small and large colonies and their analysis are presented in attached Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

On this occasion there was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values (Tables 9 and 12). However, the levels of toxicity observed in the 24 hour exposure group in the absence of metabolic activation were not as great as those observed in the preliminary toxicity test. It was once again considered that this may have been due to the presence of precipitate resulting in variable exposure of the test material to the cells or just due to the normal variations seen in biological systems. However, based on the levels of toxicity observed in the 24-hour exposure group of the preliminary toxicity test, it was considered that a slightly higher dose level would have resulted in an excessive level of toxicity. Therefore, with no evidence of any statistically significant increases in mutant frequency at any of the dose levels, including the maximum recommended dose level in the 4-hour exposure groups, in either the first or second experiment, the test material was considered to have been adequately tested. There was no evidence of any reductions in viabilities (%V), therefore indicating that residual toxicity had not occurred in either the absence or presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a modest effect on the toxicity of the test material. It should also be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in slightly higher levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test material was observed at all of the test material dose levels.

The numbers of small and large colonies and their analysis are presented in attached Tables 10 and 13.

CONCLUSION

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results (migrated information):
other: Non-mutagenic

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 312.5 to 5000 µg/ml in both the absence and presence of metabolic activation for the first experiment. For the second experiment the dose range was 78.13 to 3750 µg/ml in the absence of metabolic activation, and 312.5 to 5000 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose level used in the mutagenicity test was the maximum recommended dose level of 5000 µg/ml for the 4-hour exposure groups in both the absence and presence of metabolic activation, and limited by test material induced toxicity for the 24-hour exposure group in the absence of metabolic activation. Precipitate of test material was observed at all of the test material dose levels investigated in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion.  The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

The test was conducted at a GLP facility in accordance with adopted test guidelines. The study outcomes were presented in well-documented report format. Therefore a reliability of 1 is assigned.

Reference 5

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

Study period:

The experimental phases of the study were performed between October 28 and December 26 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: - Study performed in accordence with GLP - Study performed according to OECD Guideline 476 (In vitro mammalian cell gene mutation test) and EU Method B.17. (Mutagenicity – in vitro Mammalian Cell Gene Mutation Test)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

other: CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced, rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main study trial I (with metabolic activation) 50…300 µg/mL
Concentration range in the main study trial I (without metabolic activation) 100…600 µg/mL
Concentration range in the main study trial II (with metabolic activation) 25…300 µg/mL
Concentration range in the main study trial II (without metabolic activation) 50…600 µg/mL

Vehicle / solvent:

Culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Remarks:

In the absence of S9, ethylmethanesulfonate (EMS) was used. In the presence of S9, benzo(a)pyrene (BP) was used. See below for details.

Details on test system and experimental conditions:

Exposure period (with and without metabolic activation): 4 hours
Mutation expression time: 8 days
Determination of survival time: 16 days
Fixation time: 10 minutes

Evaluation criteria:

Assay Acceptance Criteria: A mutation assay was considered acceptable if the following criteria were met:
a. The criteria for acceptability were minimum 60% absolute cloning efficiency in negative controls (solvent used) and a spontaneous mutant frequency less than 20 per 10000000 clonable cells.
b. Positive controls induce a significant increase in the mutant frequency above the concurrent negative control.

Assay Evaluation Criteria: TOFIX-S was considered positive in the mutation assay if:
i. TOFIX-S causes a concentration-related biologically significant increase in mutant frequency in comparison with the concurrent negative control and TOFIX-S causes a three-fold increase in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases were statistically significant and outside of the laboratory historical negative (solvent used) control range.
ii. A net increase in mutant colonies of treated above the concurrent control was observed in at least two of the concentrations tested.

Statistics:

Statistical Analysis: Weighted regression analysis was performed to evaluate the dose response relationship on TOFIX-S treatment groups against negative control (excluding positive controls).

Species / strain:

other: Chinese Hamster Ovary K1 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The absolute cloning efficiency in the negative control was above 60% during both the trials. The mutant frequency, in the negative control group was less than 20 per 10000000 clonable cells during both the trials, validating the acceptability of the test system. A significant dose-related increase in the mutation frequency was not observed in any of the treated concentrations and the mutation frequency was comparable to that from the negative control group. The increased mutant frequency observed in the positive controls in trials I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Remarks on result:

other: other: HPRT locus

Remarks:

Migrated from field 'Test system'.

Conclusions:

TOFIX-S does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells both in the absence and presence of metabolic activation, under the experimental conditions as described.

Executive summary:

TOFIX-S was tested in two independent experiments, with and without metabolic activation (2% v/v S9). Ethyl methanesulfonate (0.4 µL/mL) and benzo(a)pyrene (6 µg/mL) were used as the positive control in the absence and presence of metabolic activation, respectively.

In the first mutagenicity experiment (Trial I), the CHO-K1 cells were exposed to TOFIX-S for 4 hours at test concentrations of 100, 200, 300, 400, 500, 600 µg/mL and 50, 100, 150, 200, 250, 300 µg/mL of culture media in the absence and presence of metabolic activation (2% v/v S9), respectively. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2% v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

A second trial was conducted to confirm the negative results obtained in trial I in the absence and presence of metabolic activation (2% v/v S9). The proliferating cells were exposed for a period of 4 hours to TOFIX-S at the test concentrations of 50, 100, 200, 400, 500, 600 and 25, 50, 100, 200, 250, 300 µg/mL of culture media in the absence and presence of metabolic activation system (2% v/v S9), respectively. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2% v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

No significant dose-related effect was observed at any of the treated concentrations during either trial of the experiments.

No relevant influence of the test item on pH value or osmolality was observed both in the absence and presence of metabolic activation during both

the trials.

The absolute cloning efficiency of the CHO-K1 cell line (negative control) was above 60% in both the trials. The spontaneous mutation level was (negative control) within the acceptable limit [less than 20 per 10⁶ clonable cells] in both the trials, validating the acceptability of the test system. The increased mutant frequency observed in positive controls in trial I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo was not investigated, since all in vitro tests were negative.

Justification for classification or non-classification

The substance was found to be non-mutagenic and non-clastogenic. Therefore the substance does not need to be classified for mutagenicity.