Registration Dossier
Registration Dossier
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EC number: 601-472-6 | CAS number: 117314-20-2
Genetic toxicity: in vitro
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:
- 2 015
- 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
- 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
Constituent 1
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
|
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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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