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EC number: 225-464-3 | CAS number: 4861-19-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- migrated information: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- The experimental phase of this study was performed between 26 May 2010 and 24 June 2010.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Orthophosphoric acid
- EC Number:
- 231-633-2
- EC Name:
- Orthophosphoric acid
- Cas Number:
- 7664-38-2
- IUPAC Name:
- phosphoric acid
- Details on test material:
- Sponsor's identification: PHOSPHORIC ACID TECHNICAL GRADE
Description : Clear colourless slightly viscous liquid
Purity : 85.01%
Batch number : HU10009
Date received : 05 May 2010
Expiry date : Not supplied
Storage conditions: Room temperature in the dark
Constituent 1
Method
- Target gene:
- Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
- 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:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone 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:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 10 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9 mix
Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 mix
Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix
Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix
Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix
Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
- Details on test system and experimental conditions:
- Test Procedure
Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
The positive and untreated controls were dosed using the standard plate incorporation method described in Section "Mutation Test - Experiment 1".
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. - Evaluation criteria:
- Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2008 and 2009 in Appendix 2.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 2008 and 2009 are presented in the attached Appendix 2.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation
Results and discussion
Test resultsopen allclose all
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS
Preliminary Toxicity Test
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:
With (+) or without (-)
S9-mix Strain Dose (µg/plate)
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 88 80 111 93 98 86 90 93 76 99 74
+ TA100 98 74 102 88 109 92 80 114 97 90 99
- WP2uvrA- 22 27 18 23 24 19 22 25 21 22 22
+ WP2uvrA- 19 32 30 31 22 23 25 24 22 20 23
Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.
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 or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of theS9-mix and the sensitivity of the bacterial strains. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table1 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 |
|||||
96 |
|
20 |
|
22 |
|
16 |
|
7 |
|
95 |
(93) |
21 |
(20) |
18 |
(21) |
21 |
(21) |
12 |
(11) |
88 |
|
18 |
|
22 |
|
25 |
|
13 |
|
EXPERIMENT 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
89 |
|
12 |
|
53 |
|
18 |
|
10 |
|
96 |
(97) |
18 |
(13) |
34 |
(40) |
21 |
(21) |
13 |
(11) |
107 |
|
10 |
|
32 |
|
23 |
|
11 |
|
Table 2 Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 08 June 2010 |
To: 11 June 2010 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
||||||||
- |
0 |
108 92 89 |
(96) 10.2# |
22 29 25 |
(25) 3.5 |
23 32 27 |
(27) 4.5 |
22 15 27 |
(21) 6.0 |
16 14 10 |
(13) 3.1 |
|
- |
50 |
86 99 115 |
(100) 14.5 |
26 27 27 |
(27) 0.6 |
33 29 27 |
(30) 3.1 |
14 21 14 |
(16) 4.0 |
10 15 16 |
(14) 3.2 |
|
- |
150 |
107 111 97 |
(105) 7.2 |
20 25 24 |
(23) 2.6 |
24 30 24 |
(26) 3.5 |
20 18 15 |
(18) 2.5 |
15 16 13 |
(15) 1.5 |
|
- |
500 |
98 103 102 |
(101) 2.6 |
29 21 23 |
(24) 4.2 |
32 26 30 |
(29) 3.1 |
20 19 18 |
(19) 1.0 |
12 7 15 |
(11) 4.0 |
|
- |
1500 |
103 103 117 |
(108) 8.1 |
23 20 18 |
(20) 2.5 |
27 31 26 |
(28) 2.6 |
16 14 22 |
(17) 4.2 |
10 15 10 |
(12) 2.9 |
|
- |
5000 |
111 119 112 |
(114) 4.4 |
25 19 22 |
(22) 3.0 |
29 27 22 |
(26) 3.6 |
13 21 19 |
(18) 4.2 |
12 13 11 |
(12) 1.0 |
|
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 |
5 |
2 |
0.2 |
80 |
||||||||
499 514 504 |
(506) 7.6 |
306 269 231 |
(269) 37.5 |
208 189 240 |
(212) 25.8 |
145 145 152 |
(147) 4.0 |
559 652 385 |
(532) 135.5 |
|||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
# Standard deviation
Table 3 Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 08 June 2010 |
To: 11 June 2010 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
||||||||
+ |
0 |
103 103 87 |
(98) 9.2# |
14 14 13 |
(14) 0.6 |
26 30 36 |
(31) 5.0 |
24 25 31 |
(27) 3.8 |
9 14 14 |
(12) 2.9 |
|
+ |
50 |
104 110 99 |
(104) 5.5 |
13 7 10 |
(10) 3.0 |
25 30 31 |
(29) 3.2 |
21 18 23 |
(21) 2.5 |
10 15 14 |
(13) 2.6 |
|
+ |
150 |
119 100 93 |
(104) 13.5 |
12 13 14 |
(13) 1.0 |
30 34 32 |
(32) 2.0 |
23 25 25 |
(24) 1.2 |
13 10 7 |
(10) 3.0 |
|
+ |
500 |
90 84 92 |
(89) 4.2 |
14 14 15 |
(14) 0.6 |
30 24 26 |
(27) 3.1 |
18 24 29 |
(24) 5.5 |
15 11 11 |
(12) 2.3 |
|
+ |
1500 |
104 107 100 |
(104) 3.5 |
12 9 14 |
(12) 2.5 |
35 29 29 |
(31) 3.5 |
27 23 33 |
(28) 5.0 |
10 15 9 |
(11) 3.2 |
|
+ |
5000 |
86 98 84 |
(89) 7.6 |
14 14 12 |
(13) 1.2 |
30 30 27 |
(29) 1.7 |
24 23 32 |
(26) 4.9 |
12 9 14 |
(12) 2.5 |
|
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 |
2 |
10 |
5 |
2 |
||||||||
405 389 381 |
(392) 12.2 |
219 197 229 |
(215) 16.4 |
300 323 324 |
(316) 13.6 |
202 133 128 |
(154) 41.4 |
242 273 216 |
(244) 28.5 |
|||
2AABP#
2AA 2-Aminoanthracene
BP Benzo(a)pyren
# Standard deviation
Table 4 Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 21 June 2010 |
To: 24 June 2010 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
||||||||
- |
0 |
122 107 101 |
(110) 10.8# |
18 9 15 |
(14) 4.6 |
30 21 33 |
(28) 6.2 |
32 26 20 |
(26) 6.0 |
20 19 15 |
(18) 2.6 |
|
- |
50 |
98 122 98 |
(106) 13.9 |
15 11 18 |
(15) 3.5 |
35 24 42 |
(34) 9.1 |
18 20 23 |
(20) 2.5 |
9 27 8 |
(15) 10.7 |
|
- |
150 |
84 101 110 |
(98) 13.2 |
13 15 8 |
(12) 3.6 |
33 25 31 |
(30) 4.2 |
27 16 24 |
(22) 5.7 |
12 14 18 |
(15) 3.1 |
|
- |
500 |
123 113 122 |
(119) 5.5 |
12 8 13 |
(11) 2.6 |
25 43 25 |
(31) 10.4 |
26 24 35 |
(28) 5.9 |
15 10 16 |
(14) 3.2 |
|
- |
1500 |
112 106 97 |
(105) 7.5 |
11 13 15 |
(13) 2.0 |
33 37 33 |
(34) 2.3 |
31 20 23 |
(25) 5.7 |
11 14 14 |
(13) 1.7 |
|
- |
5000 |
89 100 101 |
(97) 6.7 |
7 8 20 |
(12) 7.2 |
35 31 30 |
(32) 2.6 |
27 20 21 |
(23) 3.8 |
7 9 10 |
(9) 1.5 |
|
Positive controls
S9-Mix
- |
Name Concentration (μg/plate) No. colonies per plate |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 |
5 |
2 |
0.2 |
80 |
||||||||
507 430 484 |
(474) 39.5 |
466 276 298 |
(347) 103.9 |
317 330 301 |
(316) 14.5 |
152 157 132 |
(147) 13.2 |
2206 2210 2107 |
(2174) 58.3 |
|||
ENNG4NQO9AA#
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
# Standard deviation
Table 5 Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 21 June 2010 |
To: 24 June 2010 |
||||||||||
With or without S9-Mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
||||||||||
Base-pair substitution type |
Frameshift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
||||||||
+ |
0 |
102 107 118 |
(109) 8.2# |
16 18 20 |
(18) 2.0 |
40 34 43 |
(39) 4.6 |
42 27 24 |
(31) 9.6 |
20 11 9 |
(13) 5.9 |
|
+ |
50 |
99 108 85 |
(97) 11.6 |
11 11 13 |
(12) 1.2 |
37 37 37 |
(37) 0.0 |
26 24 32 |
(27) 4.2 |
15 19 10 |
(15) 4.5 |
|
+ |
150 |
81 82 95 |
(86) 7.8 |
11 9 22 |
(14) 7.0 |
44 42 35 |
(40) 4.7 |
37 34 22 |
(31) 7.9 |
23 13 9 |
(15) 7.2 |
|
+ |
500 |
99 108 136 |
(114) 19.3 |
19 13 16 |
(16) 3.0 |
33 36 35 |
(35) 1.5 |
30 23 26 |
(26) 3.5 |
13 7 7 |
(9) 3.5 |
|
+ |
1500 |
101 76 75 |
(84) 14.7 |
14 9 15 |
(13) 3.2 |
32 41 34 |
(36) 4.7 |
25 25 30 |
(27) 2.9 |
10 11 10 |
(10) 0.6 |
|
+ |
5000 |
93 77 84 |
(85) 8.0 |
14 9 13 |
(12) 2.6 |
40 41 41 |
(41) 0.6 |
23 31 29 |
(28) 4.2 |
9 12 10 |
(10) 1.5 |
|
Positive controls
S9-Mix
+ |
Name Concentration (μg/plate) No. colonies per plate |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 |
2 |
10 |
5 |
2 |
||||||||
592 675 673 |
(647) 47.4 |
145 132 152 |
(143) 10.1 |
299 262 321 |
(294) 29.8 |
188 198 246 |
(211) 31.0 |
157 166 161 |
(161) 4.5 |
|||
2AABP#
2AA 2-Aminoanthracene
BP Benzo(a)pyren
# Standard deviation
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Introduction.
The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods.
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.
Results.
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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
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 or exposure method.
Conclusion.
The test material was considered to be non-mutagenic under the conditions of this test.
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