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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8.10.2014-6.11.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, USA, EPA OCSPP harmonized guideline 870.5100 - Bacterial Reverse Mutation Test, Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries) and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(EC) 440/2008 of 30 May 2008
Deviations:
yes
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate is included as an attachment to the study report.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
nonatitanium(4+) tetrasodium hydrate icosaoxidandiide
EC Number:
601-472-6
Cas Number:
117314-20-2
Molecular formula:
Sodium form: Na4Ti9O20 × n H2O Sodium/hydrogen form: 50 % Na4Ti9O20 × n H2O, 50 % Na2H2Ti9O20 × n H2O
IUPAC Name:
nonatitanium(4+) tetrasodium hydrate icosaoxidandiide
Test material form:
solid: particulate/powder
Remarks:
powder
Details on test material:
- Substance type: commercial product (pure active substance)
- Physical state: Solid powder
- Storage condition of test material: Ambient temperature, humidity and pressure. Stored in sealed containers in darkness.
- Stability under test conditions: Stable
- Purity: ca 100 %
- Particle size distribution: 2.92% <100μm
- Crystal structure: TiO6-octaedra
- Density: 2.83 x 10^3 kg/m3
- pH value: the pH value of the substance in an aqueous solution is appr. 11

Method

Target gene:
Type of mutations indicated: frame shift mutations, base-pair substitutions
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: Genotype: trp-; uvrA-:; Type of mutations indicated: base-pair substitution
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Genotype: his C 3076; rfa-; uvrB-: /his D 3052; rfa-; uvrB-;R-factor: /his G 46; rfa-; uvrB-: /his G 46; rfa-; uvrB-;R-factor; Type of mutations indicated: frame shift mutations/base-pair substitutions
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile, distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
Negative (untreated) controls were used in parallel with the test item.
Negative solvent / vehicle controls:
yes
Remarks:
Culture with vehicle (sterile, distilled water)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
2-Aminoanthracene and benzo(a)pyrene were used in the series of plates with S9-mix
Details on test system and experimental conditions:
Experiment 1.
DOSES: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate were assayed in triplicate against each tester strain, using the direct plate incorporation method.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h (incubation at 37 C± 3 C)

NUMBER OF REPLICATIONS: 3

OTHER EXAMINATIONS:
All of the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

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

METHOD OF APPLICATION: preincubation

DOSES: 50, 150, 500, 1500 and 5000 ug/plate were assayed in triplicate against each tester strain, using the pre-incubation method.

DURATION
- Exposure duration: 20 min preincubation (37 C± 3 C with shaking), 48 h incubation (37 C± 3 C)

NUMBER OF REPLICATIONS: 3

OTHER EXAMINATIONS:
All of the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
The individual plate counts, the mean number of revertant colonies and the standard deviations were determined for the test item, positive and vehicle controls, both with and without metabolic activation.

The analyses were appropriate to determine the mutagenicity of the test item.

A history profile of vehicle, untreated and positive control values (reference items) are also presented.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium strains TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble in water. However, the test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.
- Precipitation: test item precipitate (greasy in appearance) was observed at and above 1500 ug/plate. This observation did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA: See table 1
Remarks on result:
other: all strains tested

Any other information on results incl. tables

Table 1. History Profile of Vehicle and Positive Control Values

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2012
Strain S9-Mix TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA pKM101 WP2pKM101
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 97 96 20 14 274 313 32 37 22 25 12 13 124 153 95 123
SD 16.9 17.9 5.1 3.3 26.8 38.2 8.0 7.8 5.8 6.7 2.7 2.8 27.0 37.4 15.9 18.0
Min 62 63 9 9 209 255 14 15 11 5 4 3 79 78 66 89
Max 162 169 39 33 321 355 58 59 48 42 24 25 216 242 161 168
Values† 780 560 561 347 20 8 668 479 898 369 880 347 64 43 56 34
POSITIVE CONTROL VALUES 2012
Strain S9-Mix TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA pKM101 WP2pKM101
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 546 1097 406 287 1033 840 734 330 167 204 778 245 764 1420 2027 633
SD 223.5 394.5 378.8 143.3 413.1 248.9 223.3 139.8 54.2 76.8 444.9 90.8 393.4 357.5 522.9 308.7
Min 266 268 85 108 541 418 256 120 81 85 146 98 158 714 1065 261
Max 3151 3241 4157 1335 1961 1284 1661 1409 420 677 3110 622 1910 2070 3071 1700
Values† 221 233 212 226 13 13 189 188 345 233 340 226 20 22 21 36
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2013
Strain S9-Mix TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA pKM101 WP2pKM101
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Total Plates 843 855 1612 798 12 6 1542 765 1655 876 1646 807 42 36 42 36
Min 68 63 9 8 191 266 15 13 10 12 5 5 110 112 105 108
Max 147 153 37 29 292 292 47 54 42 43 26 23 162 181 154 162
Mean 103 101 20 15 255 279 28 33 22 26 11 13 138 152 124 137
SD 14.4 15.6 4.4 3.5 47.3 18.4 6.6 7.1 5.0 5.1 3.1 3.5 14.4 22.2 15.4 17.1
POSITIVE CONTROL VALUES 2013
Strain S9-Mix TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA pKM101 WP2pKM101
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Total Plates 849 861 810 795 6 6 765 762 828 849 825 804 21 30 21 30
Min 240 349 91 103 668 673 129 101 102 84 113 86 501 840 302 385
Max 1429 3117 3750 1153 1016 694 1275 733 783 669 2161 1238 1124 2391 2889 1198
Mean 543 1211 644 250 842 684 611 320 207 226 813 286 698 1359 1288 665
SD 192.1 509.3 685.5 98.9 246.1 14.elo 256.3 120.9 76.9 92.6 384.2 127.7 263.1 441.9 894.7 305.7

SD Standard deviation

Min Minimum value

Max Maximum value

† Number of mean values used to create dataset

Table 2 Spontaneous Mutation Rates (Concurrent Negative Controls).

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
71 (81) 8 (15) 23 (26) 17 (14) 7 (10)
87 13 31 12 12
84 23 23 12 11

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
103 (100) 21 (15) 11 (17) 11 (14) 11 (9)
95 11 23 12 7
103 12 17 20 9

Table 3. Test Results:

Experiment 1 – Without Metabolic Activation

Test Period From: 20 October 2014 To: 23 October 2014
S9-Mix
(-)
Dose Level Per Plate Number of revertants (mean) +/- SD
Base-pair substitution strains Frameshift strains
TA100 TA1535 WP2uvrA TA98 TA1537
Solvent
Control (WATER)
87 (80) 16 (13) 27 (23) 19 (20) 9 (11)
76 6.4+ 11 2.5 20 3.5 19 1.2 13 2.1
76   13   23   21   12  
1.5 ug 82 (82) 8 (11) 28 (26) 20 (24) 9 (10)
83 0.6 16 4.6 20 4.9 27 3.5 13 2.6
82   8   29   24   8  
5 ug 75 (73) 12 (15) 28 (27) 17 (15) 5 (7)
79 7.2 19 3.8 36 9.5 12 2.5 5 4.0
65   13 17   15 12  
15 ug 107 (91) 12 (10)  24 (23) 15 (15) 8 (7)
82 14.2 11 2.6 29 6.0 17 2.5 8 1.7
83   7   17   12   5  
50 ug 84 (76) 20 (14) 19 (21) 12 (15) 11 (10)
60 13.6 15 6.0 12 10.7 12 5.2 12 2.6
83   8 33   21 7  
150 ug 83 (86) 15 (12) 11 (16) 13 (16) 9 (10)
91 4.6 15 4.6 21 5.0 24 7.0 5 5.0
83   7   17   11   15  
500 ug 86 (83) 12 (14) 12 (20) 13 (15) 5 (11)
72 9.5 20 5.7 28 8.0 15 1.5 16 5.6
90   9 20   16 12  
1500 ug 76P (80) 12P (12) 21P (17) 11P (16) 11P (12)
83P 3.5 13P 1.0 15P 3.5 20P 4.6 16P 4.0
80P   11P   15P   17P   8P  
5000 ug 100P (89) 11P (11) 15P (21) 11P (10) 15P (10)
92P 12.2 8P 3.5 21P 6.5 8P 1.7 7P 4.2
76P   15P 28P   11P 9P  
Positive controls S9-Mix (-) Name Dose Level No. of Revertants ENNG ENNG ENNG 4NQO 9AA
3 ug 5 ug 2 ug 0.2 ug 80 ug
648 (632) 859 (852) 1121 (1162) 158 (178) 970 (1186)
539 85.7 841 9.9 1276 100.4 219 35.8 1056 302.1
708   857   1088   156   1531  

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

+ Standard deviation

Experiment 1 – With Metabolic Activation

Test Period From: 20 October 2014 To: 23 October 2014
S9-Mix
(+)
Dose Level Per Plate Number of revertants (mean) +/- SD
Base-pair substitution strains Frameshift strains
TA100 TA1535 WP2uvrA TA98 TA1537
Solvent
Control (WATER)
98 (87) 17 (13) 21 (19) 16 (20) 19 (12)
79 10.0+ 12 3.2 21 2.9 24 4.0 9 5.8
83   11   16   21   9  
1.5 ug 74 (74) 9 (11) 20 (24) 16 (19) 11 (13)
76 2.0 9 3.5 24 3.5 29 9.3 15 2.1
72   15   27   11   12  
5 ug 79 (77) 11 (15) 24 (21) 16 (16) 15 (10)
86 10.7 11 7.5 20 2.6 16 0.6 7 4.2
65   24 19   15 9  
15 ug 71 (78) 11 (12)  24 (23) 19 (20) 16 (14)
72 11.3 9 3.6 25 2.6 23 2.3 16 4.0
91   16   20   19   9  
50 ug 95 (93) 12 (13) 17 (19) 12 (18) 19 (12)
82 10.1 11 2.1 20 1.5 17 6.6 11 6.1
102   15 19   25 7  
150 ug 69 (81) 12 (12) 29 (21) 15 (21) 15 (15)
107 22.2 11 0.6 13 8.0 23 4.9 19 3.5
68   12   20   24   12  
500 ug 79 (91) 13 (10) 24 (23) 21 (24) 9 (11)
83 20.8 7 3.1 19 3.2 27 3.0 15 3.8
115   11 25   24 8  
1500 ug 98P (82) 16P (14) 25P (23 17P (19) 20P (16)
64P 17.0 13P 2.1 23P 2.5 17P 3.5 16P 4.5
83P   12P   20p   23P   11P  
5000 ug 86P (82) 7P (8) 19P (16) 15P (17) 8P (10)
76P 53. 8P 1.0 13P 3.1 25P 6.8 5P 5.7
84P   9P 15P   12P 16P  
Positive controls S9-Mix
(+)
Name Dose Level No. of Revertants 2AA 2AA 2AA BP 2AA
3 ug 5 ug 2 ug 5 ug 80 ug
1431 (1544) 188 (216) 237 (237) 275 (261) 674 (677)
1612 98.5 211 30.8 237 0.0 257 12.5 659 19.7
1589   249   237   251   698  

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

+ Standard deviation

Experiment 2 – Without Metabolic Activation

Test Period From: 3 November 2014 To: 6 November 2014
S9-Mix
(-)
Dose Level Per Plate Number of revertants (mean) +/- SD
Base-pair substitution strains Frameshift strains
TA100 TA1535 WP2uvrA TA98 TA1537
Solvent
Control (WATER)
92 (101) 11 (13) 25 (21) 12 (15) 9 (11)
100 9.0+ 11 3.5 23 5.3 15 2.5 11 2.0
110   17   15   17   13  
50 ug 91 (93) 9 (13) 13 (14) 16 (13) 9 (12)
84 9.6 16 3.8 17 2.3 12 2.3 15 3.1
103   15   13   12   13  
150 ug 106 (100) 9 (9) 13 (14) 24 (16) 9 (11)
87 11.3 9 0 11 4.2 11 7.8 8 4.9
107   9 19   12 17  
500 ug 104 (103) 15 (13)  11 (15) 15 (16) 16 (9)
96 7.0 12 2.1 13 4.7 9 3.8 3 6.6
110   11   20   16   8  
1500 ug 107P (92) 13P (12) 20P (17) 11P (10) 9P (10)
88P 13.1 15P 3.1 20P 5.2 9P 1.2 8P 2.1
82P   9P 11P   11P 12P  
5000 ug 79P (80) 13P (16) 13P (16) 16P (15) 7P (8)
87P 6.1 20P 3.6 19P 3.0 11P 4.0 5P 4.2
75P   15P   16P   19P   13P  
Positive controls S9-Mix
(-)
Name Dose Level No. of Revertants ENNG ENNG ENNG 4NQO 9AA
3 ug 5 ug 2 ug 0.2 ug 80 ug
763 (660) 186 (171) 664 (644) 198 (211) 517 (441)
664 105.1 114 51.6 631 17.8 227 14.7 429 71.2
553   214   636   208   376  

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

+ Standard deviation

Experiment 2 – With Metabolic Activation

Test Period From: 3 November 2014 To: 6 November 2014
S9-Mix
(+)
Dose Level Per Plate Number of revertants (mean) +/- SD
Base-pair substitution strains Frameshift strains
TA100 TA1535 WP2uvrA TA98 TA1537
Solvent
Control (WATER)
107 (103) 9 (11) 20 (21) 23 (20) 8 (9)
96 6.4+ 12 1.7 17 4.0 16 3.5 11 1.7
107   12   25   20   8  
50 ug 118 (111) 11 (13) 17 (18) 20 (18) 13 (10)
108 6.4 12 2.1 15 3.1 16 2.1 5 4.4
106   15   21   17   12  
150 ug 100 (93) 19 (14) 19 (20) 32 (21) 8 (9)
100 12.1 12 4.4 24 4.0 17 3.8 12 2.3
79   11 16   24 8  
500 ug 116 (104) 9 (10) 25 (23) 25 (20) 19 (15)
92 12.1 9 1.2 19 3.2 27 10.4 15 3.5
104   11   24   8   12  
1500 ug 86P (93) 11P (11) 25P (20) 23P (18) 8P (9)
88P 12.0 12P 0.6 21P 5.0 13P 5.0 7P 2.6
104P   11P 15P   19P 12P  
5000 ug 100P (102) 11P (11) 23P (20) 21P (20) 9P (14)
94P 8.6 11P 0.6 25P 6.4 23P 3.1 13P 5.0
111P   12P   13P   17P   19P  
Positive controls S9-Mix
(+)
Name Dose Level No. of Revertants 2AA 2AA 2AA BP 2AA
1 ug 2 ug 10 ug 5 ug 2 ug
3626 (3505) 261 (279) 178 (187) 106 (123) 535 (562)
3417 108.8 287 15.9 178 16.2 126 16.2 516 64.5
3473   290   206   138   636  

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

+ Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative. Not classified according to the CLP Regulation based on the Reverse Mutation Assay 'Ames Test' using Salmonella
typhimurium and Escherichia coli. The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 ug/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 μg/plate.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of S9-mix, in the second mutation test (pre-incubation method). A test item precipitate (greasy in appearance) was observed at and above 1500 g/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant

colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre-incubation method).

The substance was considered to be non-mutagenic under the conditions of the test.

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