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EC number: 203-657-3 | CAS number: 109-23-9
- 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 May 2018 to 25 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
- Version / remarks:
- 24 November 2000
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. 77:33748-33749)
- Version / remarks:
- Adopted 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Envigo Research Limited, Shardlow Business Park ,Shardlow, Derbyshire, DE72 2GD, UK
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N,N'-methylenedistearamide
- EC Number:
- 203-657-3
- EC Name:
- N,N'-methylenedistearamide
- Cas Number:
- 109-23-9
- Molecular formula:
- C37H74N2O2
- IUPAC Name:
- N,N'-methylenedistearamide
Constituent 1
Method
- Target gene:
- histidine and tryptophan locus
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Microsomal fractions (CD Sprague-Dawley)
- Test concentrations with justification for top dose:
- Mutagenicity: Experiment 1 (with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).
Mutagenicity: Experiment 2 (with and without metabolic activation): 15, 50, 150, 500, 1500 and 5000 μg/plate. The dose range used for Experiment 2 was determined by the results of Experiment 1. - Vehicle / solvent:
- - Vehicle/solvent used: Dimethyl formamide (purity: 99.98%)
- Justification for choice of solvent/vehicle: The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.
Controls
- Untreated negative controls:
- yes
- Remarks:
- Untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl formamide
- 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: preincubation
DURATION
- Exposure duration: approximately 48 hours at 37°C
- Expression time: none
SELECTION METHOD:
The strains used will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium.
NUMBER OF REPLICATIONS: triplicates
METHOD:
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).
Experiment 1 – plate incorporation
The dose range for Experiment 1 was based on OECD TG 471 and was 1.5 to 5000 μg/plate.
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer (or 0,5 mL of S9-mix for the metabolic activation) and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation. Further sporadic manual counts were also performed due to spreading colonies which prevented an accurate automated count.
Experiment 2 – pre-incubation method
The experiment was repeated on a separate day 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 15 to 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology.
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer (or 0,5 mL of S9-mix for the metabolic activation) and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of 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. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation. Further sporadic manual counts were also performed due to spreading colonies which prevented an accurate automated count. - 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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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.
Acceptability criteria are listed in 'Any other information on materials incl. methods'. - 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. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported in the tables section.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA100, TA1537, TA98
- Remarks:
- Experiment 1 (plate incorporation)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- 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 (plate incorporation)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA100, TA1537, TA98
- Remarks:
- Experiment 2 (pre-incubation)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- 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 (pre-incubation)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MAIN TEST: Test results for experiment 1 can be found in Table 2,3 and experiment 2 in Table 3,4 in 'Any other information on results incl. tables'.
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: tested up to levels of precipitation (above 500 μg/plate)
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see Table 1 in ‘Any other information on results incl. tables’.
- Negative (solvent/vehicle) historical control data: see Table 1 in ‘Any other information on results incl. tables’.
Any other information on results incl. tables
Table 1. Historical positive control and vehicle/negative control values 2017
TA100
|
TA1535
|
WP2uvrA
|
TA98
|
TA1537
|
||||||
|
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Combined vehicle and untreated control values |
|
|
|
|
|
|
|
|
|
|
Values |
472 |
463 |
950 |
477 |
862 |
431 |
935 |
462 |
918 |
449 |
Mean |
92 |
92 |
18 |
17 |
25 |
31 |
22 |
26 |
13 |
14 |
SD |
14.4 |
15.4 |
6.5 |
7.1 |
7.0 |
7.4 |
6.9 |
6.8 |
3.4 |
3.4 |
|
|
|
|
|
|
|
|
|
|
|
Positive control values |
|
|
|
|
|
|
|
|
|
|
Values |
472 |
463 |
475 |
472 |
431 |
431 |
468 |
462 |
459 |
448 |
Mean |
769 |
1415 |
773 |
260 |
682 |
257 |
224 |
191 |
305 |
380 |
SD |
353.2 |
553.3 |
5.66 |
71.1 |
231.3 |
132.5 |
62.9 |
83.9 |
132.9 |
113.8 |
Table 2. Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 17 May 2018 |
To: 20 May 2018 |
|||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMF) |
150 125 118 |
(131) 16.8# |
13 18 29 |
(20) 8.2 |
30 24 20 |
(25) 5.0 |
31 15 19 |
(22) 8.3 |
15 6 8 |
(10) 4.7 |
|
1.5 µg |
116 127 133 |
(125) 8.6 |
24 34 14 |
(24) 10.0 |
29 23 22 |
(25) 3.8 |
25 24 17 |
(22) 4.4 |
10 12 7 |
(10) 2.5 |
|
5 µg |
132 96 130 |
(119) 20.2 |
18 21 13 |
(17) 4.0 |
45 25 30 |
(33) 10.4 |
22 21 18 |
(20) 2.1 |
4 11 7 |
(7) 3.5 |
|
15 µg |
109 132 119 |
(120) 11.5 |
13 14 25 |
(17) 6.7 |
19 20 27 |
(22) 4.4 |
15 22 27 |
(21) 6.0 |
4 6 16 |
(9) 6.4 |
|
50 µg |
128 108 126 |
(121) 11.0 |
16 10 9 |
(12) 3.8 |
20 20 28 |
(23) 4.6 |
16 25 26 |
(22) 5.5 |
8 8 11 |
(9) 1.7 |
|
150 µg |
105 116 150 |
(124) 23.5 |
13 10 9 |
(11) 2.1 |
28 26 32 |
(29) 3.1 |
25 32 31 |
(29) 3.8 |
5 13 24 |
(14) 9.5 |
|
500 µg |
102 P 138 P 128 P |
(123) 18.6 |
13 P 11 P 18 P |
(14) 3.6 |
22 P 39 P 34 P |
(32) 8.7 |
19 P 13 P 15 P |
(16) 3.1 |
11 P 7 P 14 P |
(11) 3.5 |
|
1500 µg |
149 P 127 P 98 P |
(125) 25.6 |
11 P 18 P 12 P |
(14) 3.8 |
30 P 21 P 23 P |
(25) 4.7 |
16 P 23 P 19 P |
(19) 3.5 |
15P 16P 25 P |
(19) 5.5 |
|
5000 µg |
103 P 99 P 118 P |
(107) 10.0 |
22 P 12 P 15 P |
(16) 5.1 |
27 P 28 P 22 P |
(26) 3.2 |
28 P 23 P 17 P |
(23) 5.5 |
12 P 14 P 13 P |
(13) 1.0 |
|
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 |
|||||||
404 549 541 |
(498) 81.5 |
866 905 708 |
(826) 104.3 |
341 389 421 |
(384) 40.3 |
206 224 253 |
(228) 23.7 |
272 439 425 |
(379) 92.6 |
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Test item precipitate
# Standard deviation
Table 3. Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 17 May 2018 |
To: 20 May 2018 |
|||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMF) |
147 125 141 |
(138) 11.4# |
26 20 18 |
(21) 4.2 |
29 29 19 |
(26) 5.8 |
19 27 25 |
(24) 4.2 |
5 8 9 |
(7) 2.1 |
|
1.5 µg |
112 95 99 |
(102) 8.9 |
22 24 23 |
(23) 1.0 |
26 14 30 |
(23) 8.3 |
26 18 21 |
(22) 4.0 |
3 8 8 |
(6) 2.9 |
|
5 µg |
150 106 114 |
(123) 23.4 |
16 26 23 |
(22) 5.1 |
23 22 36 |
(27) 7.8 |
21 28 21 |
(23) 4.0 |
3 8 6 |
(6) 2.5 |
|
15 µg |
114 138 140 |
(131) 14.5 |
12 21 17 |
(17) 4.5 |
28 22 35 |
(28) 6.5 |
24 14 35 |
(24) 10.5 |
7 10 5 |
(7) 2.5 |
|
50 µg |
153 128 134 |
(138) 13.1 |
14 10 14 |
(13) 2.3 |
27 37 34 |
(33) 5.1 |
29 15 33 |
(26) 9.5 |
8 12 11 |
(10) 2.1 |
|
150 µg |
147 139 101 |
(129) 24.6 |
10 10 23 |
(14) 7.5 |
27 23 27 |
(26) 2.3 |
19 20 32 |
(24) 7.2 |
13 10 16 |
(13) 3.0 |
|
500 µg |
83 P 93 P 102 P |
(93) 9.5 |
12 P 8 P 17 P |
(12) 4.5 |
32 P 27 P 40 P |
(33) 6.6 |
26 P 28 P 36 P |
(30) 5.3 |
7 P 15 P 19 P |
(14) 6.1 |
|
1500 µg |
124 P 17P 18P |
(109) 13.0 |
15 P 21 P 7 P |
(14) 7.0 |
33 P 29 P 30 P |
(27) 5.5 |
17 P 36 P 21 P |
(25) 10.0 |
9 P 9 P 11 P |
(10) 1.2 |
|
5000 µg |
100 P 102 P 110 P |
(104) 5.3 |
13 P 17 P 16 P |
(15) 2.1 |
30 P 30 P 24 P |
(28) 3.5 |
24 P 20 P 22 P |
(22) 2.0 |
12 P 12 P 11 P |
(12) 0.6 |
|
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 |
|||||||
2441 1766 2256 |
(2154) 348.8 |
386 360 356 |
(367) 16.3 |
217 221 183 |
(207) 20.9 |
133 130 133 |
(132) 1.7 |
371 290 333 |
(331) 40.5 |
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
P Test item precipitate
# Standard deviation
Table 4. Experiment 2 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 22 May 2018 |
To: 25 May 2018 |
|||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMF) |
104 103 107 |
(105) 2.1# |
9 8 17 |
(11) 4.9 |
21 19 26 |
(22) 3.6 |
14 18 13 |
(15) 2.6 |
13 7 5 |
(8) 4.2 |
|
15 µg |
120 116 121 |
(119) 2.6 |
12 14 19 |
(15) 3.6 |
29 25 19 |
(24) 5.0 |
15 13 15 |
(14) 1.2 |
4 6 3 |
(4) 1.5 |
|
50 µg |
105 101 92 |
(99) 6.7 |
12 9 14 |
(12) 2.5 |
25 9 15 |
(16) 8.1 |
18 13 7 |
(13) 5.5 |
12 5 3 |
(7) 4.7 |
|
150 µg |
103 111 110 |
(108) 4.4 |
12 7 18 |
(12) 5.5 |
20 21 28 |
(23) 4.4 |
17 18 16 |
(17) 1.0 |
7 8 3 |
(6) 2.6 |
|
500 µg |
105 P 109 P 97 P |
(104) 6.1 |
18 P 10 P 9 P |
(12) 4.9 |
31 P 29 P 15 P |
(25) 8.7 |
16 P 21 P 19 P |
(19) 2.5 |
4 P 10 P 16 P |
(10) 6.0 |
|
1500 µg |
86 P 98 P 97 P |
(94) 6.7 |
14 P 9 P 11 P |
(11) 2.5 |
20 P 18 P 18 P |
(19) 1.2 |
15 P 23 P 13 P |
(17) 5.3 |
6 P 5 P 8 P |
(6) 1.5 |
|
5000 µg |
89 P 88 P 91 P |
(89) 1.5 |
16 P 13 P 9 P |
(13) 3.5 |
24 P 27 P 18 P |
(23) 4.6 |
11 P 17 P 14 P |
(14) 3.0 |
8 P 10 P 12 P |
(10) 2.0 |
|
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 |
|||||||
947 811 852 |
(870) 69.8 |
687 535 665 |
(629) 82.1 |
554 543 412 |
(503) 79.0 |
160 150 130 |
(147) 15.3 |
412 356 266 |
(345) 73.7 |
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Test item precipitate
# Standard deviation
Table 5. Experiment 2 – With Metabolic Activation (Pre-Incubation)
Test Period |
From: 22 May 2018 |
To: 25 May 2018 |
|||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMF) |
118 117 125 |
(120) 4.4# |
9 10 9 |
(9) 0.6 |
39 26 37 |
(34) 7.0 |
25 19 29 |
(24) 5.0 |
10 10 18 |
(13) 4.6 |
|
15 µg |
125 111 112 |
(116) 7.8 |
7 9 13 |
(10) 3.1 |
21 36 29 |
(29) 7.5 |
21 30 27 |
(26) 4.6 |
21 12 16 |
(16) 4.5 |
|
50 µg |
120 125 134 |
(126) 7.1 |
17 11 8 |
(12) 4.6 |
28 22 28 |
(26) 3.5 |
27 25 30 |
(27) 2.5 |
6 11 11 |
(9) 2.9 |
|
150 µg |
113 118 111 |
(114) 3.6 |
9 14 18 |
(14) 4.5 |
23 29 25 |
(26) 3.1 |
21 32 26 |
(26) 5.5 |
12 14 17 |
(14) 2.5 |
|
500 µg |
131 P 121 P 111 P |
(121) 10.0 |
11 P 11 P 14 P |
(12) 1.7 |
29 P 38 P 31 P |
(33) 4.7 |
30 P 28 P 23 P |
(27) 3.6 |
19P 20P 10 P |
(16) 5.5 |
|
1500 µg |
100 P 124 P 84 P |
(103) 20.1 |
15 P 17 P 13 P |
(15) 2.0 |
19 P 16 P 14 P |
(16) 2.5 |
21 P 32 P 19 P |
(24) 7.0 |
8 P 17 P 14 P |
(13) 4.6 |
|
5000 µg |
93 P 97 P 101 P |
(97) 4.0 |
10 P 31 P 32 P |
(12) 1.5 |
30 P 21 P 23 P |
(25) 4.7 |
24 P 25 P 19 P |
(23) 3.2 |
14 P 12 P 4 P |
(10) 5.3 |
|
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 |
|||||||
1701 2056 1818 |
(1858) 180.9 |
262 207 233 |
(234) 27.5 |
156 161 163 |
(160) 3.6 |
160 175 148 |
(161) 13.5 |
310 256 262 |
(276) 29.6 |
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
P Test item precipitate
# Standard deviation
Applicant's summary and conclusion
- Conclusions:
- In this GLP compliant study, performed according to OECD 471, the test substance and its metabolites did not induce gene mutations in the tested strains of S. typhimurium and E. coli
- Executive summary:
In this GLP compliant study, performed according to OECD 471, the mutagenic potential of the test substance was evaluated in vitro inSalmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. These strains 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 (plate incorporation) was based on OECD TG 471 and was 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/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 15, 50, 150, 500, 1500 and 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology. All of the plates were incubated at 37 ± 3oC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 µg/plate because of test item precipitation. A single manual count was also performed due to spreading colonies which prevented an accurate automated count.
The vehicle (dimethyl formamide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.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). No biologically relevant 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). Minor statistical values were noted in Experiment 2 (TA100 at 15 μg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. In conclusion, the test substance was considered to be non-mutagenic under the conditions of this test.
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