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EC number: 600-548-6 | CAS number: 1042662-40-7
- 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:
- Experimental start date 30 January 2017 Experimental completion date 03 March 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
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',5'-di-tert-butyl-3-{5-tert-butyl-2-[3-(4-tert-butyl-2-{3',5'-di-tert-butyl-2-hydroxy-5-methyl-[1,1'-biphenyl]-3-yl}phenoxy)propoxy]phenyl}-5-methyl-[1,1'-biphenyl]-2-ol
- EC Number:
- 600-548-6
- Cas Number:
- 1042662-40-7
- Molecular formula:
- C65H84O4
- IUPAC Name:
- 3',5'-di-tert-butyl-3-{5-tert-butyl-2-[3-(4-tert-butyl-2-{3',5'-di-tert-butyl-2-hydroxy-5-methyl-[1,1'-biphenyl]-3-yl}phenoxy)propoxy]phenyl}-5-methyl-[1,1'-biphenyl]-2-ol
Constituent 1
- Specific details on test material used for the study:
- Identification: 2,2’-[propane-1,3-diylbis(oxy)]bis(3”,5,5”-tri-tert-butyl-5’-methyl-1,1’:3’,1”-terphenyl-2’-ol)
CAS Number: 1042662-40-7
Physical state/Appearance: White powder
Batch: 1502501005
Purity: 100%
Expiry Date: 01 February 2025
Storage Conditions: Room temperature in the dark
Method
- Target gene:
- Histidine operon for Salmonellla
Tryptophan operon for Escherichia
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver microsomal preparation (S9-mix)
- Test concentrations with justification for top dose:
- Esperiment 1 - With and without metabolic activation
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 - With and without metabolic activation
15, 50, 150, 500, 1500, 5000 µg/plate - Vehicle / solvent:
- Dimethyl formamide
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rate
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- (-S9 mix) 2 µg/plate fpr WP2uvrA, 3 µg/plate for TA 100 and 5 µg/plate for TA1535
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- (-S9 mix) 80 µg/plate for TA1537
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- (-S9 mix) 0.2 µg/plate for TA98
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA)
- Remarks:
- (+ S9 mix) 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- (+ S9 mix) 5 µg/plate for TA98
- Details on test system and experimental conditions:
- Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. 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.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
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 and above 1500 µg/plate because of test item precipitation.
Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as 15 to 5000 µg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Without Metabolic Activation
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. 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.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
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 again performed at and above 1500 µg/plate because of test item precipitation. - Rationale for test conditions:
- In solubility checks performed in house, the test item was noted as insoluble in sterile distilled water, acetonitrile and dimethyl sulphoxide at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in dimethyl formamide at 50 mg/mL and tetrahydrofuran at 200 mg/mL. Dimethyl formamide was selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 30 minutes at 40 °C on the day of each experiment. No correction for purity was required. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. - 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 results
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- 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. 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 item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 (plate incorporation) and Table 4 and Table 5 for Experiment 2 (pre-incubation).
A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.
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 (5000 µg/plate) was selected in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains (except TA100 dosed in the absence of S9-mix) at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Slightly weakened bacterial background lawns were noted to TA100 in the absence of S9-mix only at 5000 µg/plate after the incorporation of the pre-incubation modification in the second mutation test. A test item precipitate (powdery in appearance) was noted at and above 500 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 metabolic activation (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 metabolic activation (S9-mix) in Experiment 2 (pre incubation method).
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 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
Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 16 February 2017 |
To: 19 February 2017 |
|||||||||||||||||||||
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) |
98 83 69 |
(83) 14.5# |
24 31 29 |
(28) 3.6 |
19 27 15 |
(20) 6.1 |
20 20 15 |
(18) 2.9 |
8 10 7 |
(8) 1.5 |
|||||||||||||
1.5 µg |
83 88 96 |
(89) 6.6 |
18 9 16 |
(14) 4.7 |
21 21 13 |
(18) 4.6 |
21 12 22 |
(18) 5.5 |
10 11 9 |
(10) 1.0 |
|||||||||||||
5 µg |
74 80 78 |
(77) 3.1 |
33 7 18 |
(19) 13.1 |
16 15 22 |
(18) 3.8 |
20 16 8 |
(15) 6.1 |
11 12 10 |
(11) 1.0 |
|||||||||||||
15 µg |
68 100 99 |
(89) 18.2 |
17 16 20 |
(18) 2.1 |
19 19 12 |
(17) 4.0 |
14 22 22 |
(19) 4.6 |
7 17 8 |
(11) 5.5 |
|||||||||||||
50 µg |
75 87 76 |
(79) 6.7 |
12 20 20 |
(17) 4.6 |
16 24 29 |
(23) 6.6 |
33 9 20 |
(21) 12.0 |
9 13 22 |
(15) 6.7 |
|||||||||||||
150 µg |
91 94 79 |
(88) 7.9 |
21 24 23 |
(23) 1.5 |
7 18 12 |
(12) 5.5 |
30 14 23 |
(22) 8.0 |
9 10 9 |
(9) 0.6 |
|||||||||||||
500 µg |
111 P 84 P 93 P |
(96) 13.7 |
16 P 15 P 31 P |
(21) 9.0 |
20 P 16 P 20 P |
(19) 2.3 |
23 P 16 P 29 P |
(23) 6.5 |
10 P 10 P 17 P |
(12) 4.0 |
|||||||||||||
1500 µg |
75 P 85 P 75 P |
(78) 5.8 |
16 P 24 P 16 P |
(19) 4.6 |
19 P 17 P 18 P |
(18) 1.0 |
22 P 24 P 24 P |
(23) 1.2 |
10 P 9 P 8 P |
(9) 1.0 |
|||||||||||||
5000 µg |
72 P 91 P 90 P |
(84) 10.7 |
20 P 13 P 16 P |
(16) 3.5 |
19 P 26 P 21 P |
(22) 3.6 |
19 P 28 P 26 P |
(24) 4.7 |
7 P 7 P 10 P |
(8) 1.7 |
|||||||||||||
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 |
|||||||||||||||||||
353 319 300 |
(324) 26.9 |
137 149 220 |
(169) 44.9 |
489 524 417 |
(477) 54.6 |
129 134 134 |
(132) 2.9 |
257 339 332 |
(309) 45.5 |
||||||||||||||
ENNG4NQO9AAP#
Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)
Test Period |
From: 16 February 2017 |
To: 19 February 2017 |
|||||||||||||||||||||
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) |
87 72 120 |
(93) 24.6# |
10 7 10 |
(9) 1.7 |
29 17 22 |
(23) 6.0 |
13 15 24 |
(17) 5.9 |
12 15 6 |
(11) 4.6 |
|||||||||||||
1.5 µg |
75 96 105 |
(92) 15.4 |
6 9 6 |
(7) 1.7 |
19 32 32 |
(28) 7.5 |
23 14 24 |
(20) 5.5 |
12 8 14 |
(11) 3.1 |
|||||||||||||
5 µg |
105 113 91 |
(103) 11.1 |
8 8 17 |
(11) 5.2 |
28 16 21 |
(22) 6.0 |
24 20 24 |
(23) 2.3 |
14 11 15 |
(13) 2.1 |
|||||||||||||
15 µg |
120 100 92 |
(104) 14.4 |
12 9 25 |
(15) 8.5 |
26 28 21 |
(25) 3.6 |
14 24 22 |
(20) 5.3 |
14 12 18 |
(15) 3.1 |
|||||||||||||
50 µg |
113 104 103 |
(107) 5.5 |
19 11 18 |
(16) 4.4 |
21 23 15 |
(20) 4.2 |
13 18 20 |
(17) 3.6 |
6 11 17 |
(11) 5.5 |
|||||||||||||
150 µg |
105 104 101 |
(103) 2.1 |
7 11 7 |
(8) 2.3 |
27 34 27 |
(29) 4.0 |
18 25 15 |
(19) 5.1 |
11 10 17 |
(13) 3.8 |
|||||||||||||
500 µg |
76 P 111 P 95 P |
(94) 17.5 |
25 P 11 P 7 P |
(14) 9.5 |
31 P 26 P 26 P |
(28) 2.9 |
18 P 16 P 23 P |
(19) 3.6 |
12 P 12 P 15 P |
(13) 1.7 |
|||||||||||||
1500 µg |
83 P 90 P 96 P |
(90) 6.5 |
8 P 9 P 12 P |
(10) 2.1 |
19 P 23 P 20 P |
(21) 2.1 |
17 P 14 P 19 P |
(17) 2.5 |
8 P 6 P 12 P |
(9) 3.1 |
|||||||||||||
5000 µg |
90 P 75 P 70 P |
(78) 10.4 |
15 P 13 P 14 P |
(14) 1.0 |
17 P 22 P 21 P |
(20) 2.6 |
17 P 12 P 14 P |
(14) 2.5 |
13 P 13 P 8 P |
(11) 2.9 |
|||||||||||||
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 |
|||||||||||||||||||
1865 1945 1919 |
(1910) 40.8 |
271 252 259 |
(261) 9.6 |
237 202 196 |
(212) 22.1 |
96 123 132 |
(117) 18.7 |
178 207 162 |
(182) 22.8 |
||||||||||||||
2AABPP#
Test Results: Experiment 2 – Without Metabolic Activation
(Pre-Incubation)
Test Period |
From: 28 February 2017 |
To: 03 March 2017 |
||||||||||
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) |
85 80 69 |
(78) 8.2# |
13 17 14 |
(15) 2.1 |
23 32 20 |
(25) 6.2 |
18 15 15 |
(16) 1.7 |
7 14 14 |
(12) 4.0 |
||
15 µg |
71 81 86 |
(79) 7.6 |
19 18 8 |
(15) 6.1 |
25 31 39 |
(32) 7.0 |
10 11 11 |
(11) 0.6 |
13 19 9 |
(14) 5.0 |
||
50 µg |
73 75 67 |
(72) 4.2 |
19 15 12 |
(15) 3.5 |
25 36 19 |
(27) 8.6 |
12 18 18 |
(16) 3.5 |
12 14 24 |
(17) 6.4 |
||
150 µg |
67 67 75 |
(70) 4.6 |
13 11 11 |
(12) 1.2 |
31 24 28 |
(28) 3.5 |
12 10 13 |
(12) 1.5 |
12 13 13 |
(13) 0.6 |
||
500 µg |
72 P 68 P 71 P |
(70) 2.1 |
10 P 11 P 19 P |
(13) 4.9 |
38 P 37 P 21 P |
(32) 9.5 |
16 P 17 P 18 P |
(17) 1.0 |
11 P 14 P 7 P |
(11) 3.5 |
||
1500 µg |
64 P 65 P 73 P |
(67) 4.9 |
12 P 8 P 8 P |
(9) 2.3 |
27 P 31 P 32 P |
(30) 2.6 |
10 P 16 P 12 P |
(13) 3.1 |
12 P 11 P 8 P |
(10) 2.1 |
||
5000 µg |
66 PS 65 PS 50 PS |
(60) 9.0 |
12 P 10 P 7 P |
(10) 2.5 |
17 P 17 P 24 P |
(19) 4.0 |
19 P 18 P 14 P |
(17) 2.6 |
20 P 13 P 11 P |
(15) 4.7 |
||
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 |
||||||||
1295 1677 1576 |
(1516) 197.9 |
1879 1910 1911 |
(1900) 18.2 |
495 562 473 |
(510) 46.4 |
223 269 211 |
(234) 30.6 |
199 168 213 |
(193) 23.0 |
|||
ENNG4NQO9AASP #
Test Results: Experiment 2 - With Metabolic Activation (Pre-Incubation)
Test Period |
From: 28 February 2017 |
To: 03 March 2017 |
||||||||||
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) |
107 85 102 |
(98) 11.5# |
15 10 10 |
(12) 2.9 |
28 26 37 |
(30) 5.9 |
15 19 12 |
(15) 3.5 |
16 18 12 |
(15) 3.1 |
||
15 µg |
82 122 100 |
(101) 20.0 |
12 9 20 |
(14) 5.7 |
30 36 37 |
(34) 3.8 |
14 16 9 |
(13) 3.6 |
10 18 12 |
(13) 4.2 |
||
50 µg |
114 92 87 |
(98) 14.4 |
16 7 14 |
(12) 4.7 |
32 27 34 |
(31) 3.6 |
23 14 18 |
(18) 4.5 |
23 15 16 |
(18) 4.4 |
||
150 µg |
80 105 88 |
(91) 12.8 |
14 9 15 |
(13) 3.2 |
39 26 43 |
(36) 8.9 |
27 16 26 |
(23) 6.1 |
17 15 19 |
(17) 2.0 |
||
500 µg |
82 P 83 P 90 P |
(85) 4.4 |
15 P 10 P 14 P |
(13) 2.6 |
21 P 38 P 40 P |
(33) 10.4 |
14 P 19 P 18 P |
(17) 2.6 |
21 P 18 P 10 P |
(16) 5.7 |
||
1500 µg |
93 P 92 P 93 P |
(93) 0.6 |
11 P 8 P 8 P |
(9) 1.7 |
33 P 37 P 37 P |
(36) 2.3 |
17 P 15 P 18 P |
(17) 1.5 |
15 P 10 P 7 P |
(11) 4.0 |
||
5000 µg |
88 P 64 P 99 P |
(84) 17.9 |
13 P 9 P 12 P |
(11) 2.1 |
29 P 31 P 29 P |
(30) 1.2 |
18 P 11 P 12 P |
(14) 3.8 |
8 P 5 P 10 P |
(8) 2.5 |
||
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 |
||||||||
1016 890 914 |
(940) 66.9 |
289 276 246 |
(270) 22.1 |
215 214 256 |
(228) 24.0 |
126 155 123 |
(135) 17.7 |
309 324 312 |
(315) 7.9 |
|||
BP2AAP
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Test item precipitate
# Standard deviation
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
P Test item precipitate
# Standard deviation
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparsebacterial background lawn
P Test Item precipitate
# Standard deviation
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
P Test Item precipitate
Applicant's summary and conclusion
- Conclusions:
- 2,2’-[propane-1,3-diylbis(oxy)]bis(3”,5,5”-tri-tert-butyl-5’-methyl-1,1’:3’,1”-terphenyl-2’-ol) was considered to be non-mutagenic under the conditions of this test.
- Executive summary:
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 typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere 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 was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item following the change in test methodology.
Results
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 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 (5000 µg/plate) was selected in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawns of any of the tester strains (except TA100 dosed in the absence of S9-mix) at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Slightly weakened bacterial background lawns were noted to TA100 in the absence of S9-mix only at 5000 µg/plate after the incorporation of the pre-incubation modification in the second mutation test. A test item precipitate (powdery in appearance) was noted at and above 500 mg/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 metabolic activation (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 metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method).
Conclusion
2,2’-[propane-1,3-diylbis(oxy)]bis(3”,5,5”-tri-tert-butyl-5’-methyl-1,1’:3’,1”-terphenyl-2’-ol)was considered to be non-mutagenic under the conditions of this test.
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