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EC number: 824-801-7 | CAS number: 1093628-27-3
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Reverse mutation assay
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was conducted between 20 July 2016 and 12 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
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:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 3-amino-N,N-dimethylpropan-1-aminium 2-C10-13-alkyl benzenesulfonate
- EC Number:
- 824-801-7
- Cas Number:
- 1093628-27-3
- Molecular formula:
- not applicable
- IUPAC Name:
- 3-amino-N,N-dimethylpropan-1-aminium 2-C10-13-alkyl benzenesulfonate
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- Identification: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001)
Physical state/Appearance: Dark amber coloured liquid
Batch: 2629-70-10
Purity: 59.7%
Expiry Date: 18 January 2019
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (40.3%) of the test item.
Method
- Target gene:
- Histidine gene
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- Experiment one (all bacterial strains) 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two
All Salmonella strains (absence of S9), Salmonella strains TA100 and TA1537 (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
E.coli strain WP2uvrA (presence and absence of S9), Salmonella strains TA1535 and TA98 (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Up to nine test item concentrations were selected per bacterial strain in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation - Vehicle / solvent:
- Dimethyl sulphoxide
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house
Controls
- Untreated negative controls:
- yes
- Remarks:
- spontaneous mutation rates (all test strains)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- dimethyl sulphoxide
- 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 (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation).
Experiment 2 included a preincubation period (0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates)
DURATION
- Preincubation period: 20 minutes (Experiment 2 only)
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): histidine deficient medium
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity) - Evaluation criteria:
- Acceptability Criteria:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls).
All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with orwithout metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
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:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Experiment 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix), in experiment 1
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Experiment 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate and in presence of S9-mix from 500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Experiment 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Experiment 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate and in presence of S9-mix from 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Experiment 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Experiment 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate and in presence of S9-mix from 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Experiment 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Experiment 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate and in the presence of S9-mix from 500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Experiment 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Experiment 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Small, statistically significant increases in revertant colony frequency were observed in the absence of S9-mix at 1.5 and 5 µg/plate. These increases were considered to be of no biological relevance according to the evaluation criteria
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weakened bacterial background lawns noted in the absence of S9-mix from 5000 µg/plate and in the presence of S9-mix from 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- 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 the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented (see any other information on results incl. tables section) and were considered to be acceptable.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented (see any other information on results incl. tables section).
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level or toxic limit, depending on bacterial strain type and presence or absence of S9-mix, was used in the second mutation test. The test item induced a stronger toxic response after the introduction of the pre-incubation method in the second mutation test with weakened bacterial background lawns noted in the absence of S9-mix from
50 µg/plate (TA100), 150 µg/plate (TA1535 and TA1537), 500 µg/plate (TA98) and at
5000 µg/plate (WP2uvrA). In the presence of S9-mix, toxicity was initially noted from
500 µg/plate (TA100 and TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate in the first mutation test (plate incorporation method) and at 5000 µg/plate in the second mutation test (pre-incubation method). These observations 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically 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 metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the absence of S9-mix at 1.5 and 5 µg/plate in the second mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle control.
The vehicle (dimethyl sulphoxide) 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.
Any other information on results incl. tables
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 |
|||||
62 |
|
13 |
|
29 |
|
18 |
|
9 |
|
76 |
(66) |
11 |
(12) |
25 |
(22) |
12 |
(17) |
15 |
(14) |
60 |
|
12 |
|
11 |
|
21 |
|
19 |
|
102 |
|
|
|||||||
124 |
(114)† |
||||||||
117 |
|
Experiment 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
68 |
|
10 |
|
17 |
|
24 |
|
7 |
|
79 |
(74) |
9 |
(10) |
11 |
(14) |
15 |
(18) |
8 |
(7) |
75 |
|
10 |
|
13 |
|
14 |
|
5 |
|
† Experimental procedure repeated at a later date (without S9-mix) due to poor colony frequency in the original test
Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 21 July 2016 26 July 2016† |
To: 24 July 2016 29 July 2016† |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100† |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
117 91 105 |
(104) 13.0# |
9 17 10 |
(12) 4.4 |
25 21 21 |
(22) 2.3 |
29 16 20 |
(22) 6.7 |
9 8 12 |
(10) 2.1 |
||
1.5 µg |
114 90 95 |
(100) 12.7 |
16 10 7 |
(11) 4.6 |
32 22 24 |
(26) 5.3 |
12 22 16 |
(17) 5.0 |
5 13 11 |
(10) 4.2 |
||
5 µg |
93 89 91 |
(91) 2.0 |
10 10 23 |
(14) 7.5 |
20 32 27 |
(26) 6.0 |
14 13 11 |
(13) 1.5 |
13 12 9 |
(11) 2.1 |
||
15 µg |
104 68 123 |
(98) 27.9 |
9 10 14 |
(11) 2.6 |
14 15 17 |
(15) 1.5 |
11 22 13 |
(15) 5.9 |
7 18 11 |
(12) 5.6 |
||
50 µg |
81 81 107 |
(90) 15.0 |
19 10 18 |
(16) 4.9 |
13 21 22 |
(19) 4.9 |
17 9 15 |
(14) 4.2 |
19 16 13 |
(16) 3.0 |
||
150 µg |
90 79 92 |
(87) 7.0 |
15 7 19 |
(14) 6.1 |
18 14 17 |
(16) 2.1 |
25 13 13 |
(17) 6.9 |
8 10 12 |
(10) 2.0 |
||
500 µg |
61 S 60 S 71 S |
(64) 6.1 |
6 10 8 |
(8) 2.0 |
13 16 9 |
(13) 3.5 |
15 16 13 |
(15) 1.5 |
11 S 4 S 10 S |
(8) 3.8 |
||
1500 µg |
22 SP 25 SP 24 SP |
(24) 1.5 |
4 SP 5 SP 3 SP |
(4) 1.0 |
7 P 7 P 8 P |
(7) 0.6 |
5 SP 2 SP 3 SP |
(3) 1.5 |
0 VP 0 VP 0 VP |
(0) 0.0 |
||
5000 µg |
0 VP 0 VP 0 VP |
(0) 0.0 |
0 VP 0 VP 0 VP |
(0) 0.0 |
3 P 3 P 3 P |
(3) 0.0 |
0 TP 0 TP 0 TP |
(0) 0.0 |
0 TP 0 TP 0 TP |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
393 408 327 |
(376) 43.1 |
253 235 306 |
(265) 36.9 |
599 551 609 |
(586) 31.0 |
229 194 217 |
(213) 17.8 |
324 361 490 |
(392) 87.1 |
† Experimental procedure repeated at a later date due to poor colony frequency in the original test
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
P Test Item precipitate
# Standard deviation
Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 21 July 2016 |
To: 24 July 2016 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
98 71 61 |
(77) 19.1# |
8 9 13 |
(10) 2.6 |
30 13 28 |
(24) 9.3 |
23 21 24 |
(23) 1.5 |
19 17 9 |
(15) 5.3 |
||
1.5 µg |
76 77 72 |
(75) 2.6 |
10 11 7 |
(9) 2.1 |
38 30 23 |
(30) 7.5 |
18 21 16 |
(18) 2.5 |
22 17 7 |
(15) 7.6 |
||
5 µg |
72 60 63 |
(65) 6.2 |
10 10 7 |
(9) 1.7 |
21 10 35 |
(22) 12.5 |
17 22 14 |
(18) 4.0 |
16 22 7 |
(15) 7.5 |
||
15 µg |
67 63 61 |
(64) 3.1 |
7 7 9 |
(8) 1.2 |
15 15 13 |
(14) 1.2 |
29 22 29 |
(27) 4.0 |
10 19 10 |
(13) 5.2 |
||
50 µg |
67 80 73 |
(73) 6.5 |
7 8 10 |
(8) 1.5 |
33 20 16 |
(23) 8.9 |
12 25 24 |
(20) 7.2 |
9 10 7 |
(9) 1.5 |
||
150 µg |
61 76 62 |
(66) 8.4 |
10 9 11 |
(10) 1.0 |
21 40 20 |
(27) 11.3 |
15 21 29 |
(22) 7.0 |
10 6 19 |
(12) 6.7 |
||
500 µg |
44 51 42 |
(46) 4.7 |
5 6 2 |
(4) 2.1 |
31 20 11 |
(21) 10.0 |
24 15 16 |
(18) 4.9 |
4 10 6 |
(7) 3.1 |
||
1500 µg |
23 SP 34 SP 27 SP |
(28) 5.6 |
3 P 4 P 6 P |
(4) 1.5 |
5 P 16 P 19 P |
(13) 7.4 |
11 P 7 P 15 P |
(11) 4.0 |
0 VP 0 VP 0 VP |
(0) 0.0 |
||
5000 µg |
0 VP 0 VP 0 VP |
(0) 0.0 |
0 VP 0 VP 0 VP |
(0) 0.0 |
10 P 9 P 5 P |
(8) 2.6 |
0 VP 0 VP 0 VP |
(0) 0.0 |
0 TP 0 TP 0 TP |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
611 646 636 |
(631) 18.0 |
225 219 197 |
(214) 14.7 |
384 460 429 |
(424) 38.2 |
96 129 126 |
(117) 18.2 |
271 277 248 |
(265) 15.3 |
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
P Test Item precipitate
# Standard deviation
Experiment 2 – Without Metabolic Activation
Test Period |
From: 09 August 2016 |
To: 12 August 2016 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
83 69 64 |
(72) 9.8# |
9 9 9 |
(9) 0.0 |
15 15 13 |
(14) 1.2 |
15 14 21 |
(17) 3.8 |
7 4 5 |
(5) 1.5 |
||
0.5 µg |
72 73 67 |
(71) 3.2 |
7 9 9 |
(8) 1.2 |
14 13 10 |
(12) 2.1 |
13 15 16 |
(15) 1.5 |
5 9 6 |
(7) 2.1 |
||
1.5 µg |
67 65 84 |
(72) 10.4 |
8 9 12 |
(10) 2.1 |
19 23 19 |
* (20) 2.3 |
16 12 14 |
(14) 2.0 |
10 3 9 |
(7) 3.8 |
||
5 µg |
83 66 75 |
(75) 8.5 |
12 8 9 |
(10) 2.1 |
23 17 26 |
** (22) 4.6 |
9 14 14 |
(12) 2.9 |
5 3 8 |
(5) 2.5 |
||
15 µg |
72 60 63 |
(65) 6.2 |
10 9 4 |
(8) 3.2 |
14 11 13 |
(13) 1.5 |
16 23 19 |
(19) 3.5 |
4 8 8 |
(7) 2.3 |
||
50 µg |
58 S 47 S 60 S |
(55) 7.0 |
6 9 6 |
(7) 1.7 |
15 16 23 |
(18) 4.4 |
19 16 20 |
(18) 2.1 |
6 7 5 |
(6) 1.0 |
||
150 µg |
49 S 63 S 48 S |
(53) 8.4 |
6 S 3 S 6 S |
(5) 1.7 |
7 11 9 |
(9) 2.0 |
12 13 13 |
(13) 0.6 |
7 S 3 S 4 S |
(5) 2.1 |
||
500 µg |
49 S 47 S 44 S |
(47) 2.5 |
2 S 4 S 3 S |
(3) 1.0 |
10 10 10 |
(10) 0.0 |
16 S 9 S 7 S |
(11) 4.7 |
3 S 2 S 2 S |
(2) 0.6 |
||
1500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
14 13 12 |
(13) 1.0 |
6 S 5 S 3 S |
(5) 1.5 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
N/T |
N/T |
9 SP 10 SP 6 SP |
(8) 2.1 |
N/T |
N/T |
||||||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
880 1191 1060 |
(1044) 156.1 |
1604 1786 1856 |
(1749) 130.1 |
356 606 532 |
(498) 128.4 |
223 198 224 |
(215) 14.7 |
109 117 173 |
(133) 34.9 |
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
N/T Not tested at this dose level
S Sparse bacterial background lawn
V Very weak bacterial background lawn
P Test Item precipitate
* p≤0.05
** p≤0.01
# Standard deviation
Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 09 August 2016 |
To: 12 August 2016 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
73 67 84 |
(75) 8.6# |
11 8 11 |
(10) 1.7 |
19 19 14 |
(17) 2.9 |
22 16 17 |
(18) 3.2 |
8 8 8 |
(8) 0.0 |
||
0.5 µg |
80 70 70 |
(73) 5.8 |
11 9 8 |
(9) 1.5 |
15 14 15 |
(15) 0.6 |
17 15 12 |
(15) 2.5 |
13 11 13 |
(12) 1.2 |
||
1.5 µg |
65 58 80 |
(68) 11.2 |
10 10 10 |
(10) 0.0 |
17 13 21 |
(17) 4.0 |
23 23 19 |
(22) 2.3 |
6 7 7 |
(7) 0.6 |
||
5 µg |
65 68 58 |
(64) 5.1 |
10 11 8 |
(10) 1.5 |
20 14 14 |
(16) 3.5 |
13 19 22 |
(18) 4.6 |
5 10 5 |
(7) 2.9 |
||
15 µg |
73 60 55 |
(63) 9.3 |
10 7 7 |
(8) 1.7 |
25 16 17 |
(19) 4.9 |
17 16 16 |
(16) 0.6 |
6 1 7 |
(5) 3.2 |
||
50 µg |
58 83 62 |
(68) 13.4 |
12 9 7 |
(9) 2.5 |
13 12 19 |
(15) 3.8 |
15 12 25 |
(17) 6.8 |
3 6 3 |
(4) 1.7 |
||
150 µg |
45 48 60 |
(51) 7.9 |
9 2 5 |
(5) 3.5 |
9 19 15 |
(14) 5.0 |
14 11 22 |
(16) 5.7 |
8 5 6 |
(6) 1.5 |
||
500 µg |
63 S 33 S 66 S |
(54) 18.2 |
5 S 5 S 3 S |
(4) 1.2 |
14 15 14 |
(14) 0.6 |
20 9 13 |
(14) 5.6 |
5 4 4 |
(4) 0.6 |
||
1500 µg |
0 V 0 V 0 V |
(0) 0.0 |
1 S 3 S 0 S |
(1) 1.5 |
11 24 14 |
(16) 6.8 |
15 S 10 S 4 S |
(10) 5.5 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
N/T |
0 VP 0 VP 0 VP |
(0) 0.0 |
1 SP 8 SP 7 SP |
(5) 3.8 |
0 VP 0 VP 0 VP |
(0) 0.0 |
N/T |
||||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
911 813 902 |
(875) 54.2 |
203 200 203 |
(202) 1.7 |
127 118 112 |
(119) 7.5 |
181 178 175 |
(178) 3.0 |
207 172 224 |
(201) 26.5 |
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
N/T Not tested at this dose level
S Sparse bacterial background lawn
V Very weak bacterial background lawn
P Test Item precipitate
# Standard deviation
Applicant's summary and conclusion
- Conclusions:
- Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001) was considered to be non-mutagenic under the conditions of this test.
- Executive summary:
Introduction
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, 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 OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
Methods
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with 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 mg/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 ranged between 0.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.
Up to nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the test item following the change in test methodology.
Results
The vehicle (dimethyl sulphoxide) 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. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level or toxic limit, depending on bacterial strain type and presence or absence of S9-mix, was used in the second mutation test. The test item induced a stronger toxic response after the introduction of the pre-incubation method in the second mutation test with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA100), 150 µg/plate (TA1535 and TA1537), 500 µg/plate (TA98) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix, toxicity was initially noted from
500 µg/plate (TA100 and TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate in the first mutation test (plate incorporation method) and at 5000 µg/plate in the second mutation test (pre-incubation method). These observations 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically 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 metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). Small, statistically significant increases in WP2uvrArevertant colony frequency were observed in the absence of S9-mix at 1.5 and 5 µg/plate in the second mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle control.
Conclusion
Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001)was considered to be non-mutagenic under the conditions of this test.
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