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EC number: 289-861-3 | CAS number: 90028-68-5 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Evernia prunastri, Usneaceae.
- 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:
- 19 July - 25 August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD Guideline 471 without any deviation
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)
- Version / remarks:
- dated 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- dated August 1998
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Methyl 2,4-dihydroxy-3,6-dimethylbenzoate
- EC Number:
- 225-193-0
- EC Name:
- Methyl 2,4-dihydroxy-3,6-dimethylbenzoate
- Cas Number:
- 4707-47-5
- Molecular formula:
- C10H12O4
- IUPAC Name:
- methyl 2,4-dihydroxy-3,6-dimethylbenzoate
- Reference substance name:
- [1R-(1α,4aβ,10aα)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid
- EC Number:
- 217-102-8
- EC Name:
- [1R-(1α,4aβ,10aα)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid
- Cas Number:
- 1740-19-8
- Molecular formula:
- C20H28O2
- IUPAC Name:
- [1R-(1α,4aβ,10aα)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid
- Reference substance name:
- 2,6-dihydroxy-4-methylbenzaldehyde
- Cas Number:
- 526-37-4
- Molecular formula:
- C8H8O3
- IUPAC Name:
- 2,6-dihydroxy-4-methylbenzaldehyde
- Reference substance name:
- 3-Chloro-2,6-dihydroxy-4-methylbenzaldehyde
- Cas Number:
- 57074-21-2
- Molecular formula:
- C8H7ClO3
- IUPAC Name:
- 3-Chloro-2,6-dihydroxy-4-methylbenzaldehyde
- Test material form:
- other: brown, viscous, sticky paste
- Details on test material:
- Name OAKMOSS ABSOLUTE
Batch no. A17 019-2
Appearance Brown, viscous, sticky paste
CAS No. 90028-68-5
EINECS-No. 289-861-3
Production date 19. Jan .2017
Expiry date 18. Jan. 2019
Storage Room temperature (20 ± 5°C), Keep away from light and humidity, keep under inert gas
Constituent 1
Constituent 2
Constituent 3
Constituent 4
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: A17 019-2
- Expiration date of the lot/batch: 24 January 2019
- Purity test date: January 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at approximately 40°C on the day of each experiment. No correction was required for purity. 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.
Method
- Target gene:
- histidine locus for Salmonella strains and tryptophan for E. coli strain
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- 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 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration and in acetone at 100 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours
CONTROLS:
- Vehicle/solvent control: DMSO
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).
NUMBER OF REPLICATIONS: Triplicate
- OTHER: 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 a test item film. Several further manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count. - Rationale for test conditions:
- The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated using the same dose range on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of 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.
- Evaluation criteria:
- Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- 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:
- None
Results and discussion
Test results
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- refer tables 7.6.1/2 to 7.6.1/5
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item induced film (light yellow in colour) was noted at and above 1500 g/plate, this observation did not prevent the scoring of revertant colonies.
MUTAGENICITY
- 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.
- In the first experiment (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns in all of the Salmonella tester strains only. In the absence of S9-mix, toxicity was observed at and above 1500 µg/plate (TA100 and TA1537) and at 5000 µg/plate (TA1535 and TA98). In the presence of S9-mix, toxicity was observed to all of the Salmonella strains at 5000 µg/plate only. No toxicity was observed in the E.coli strain, WP2uvrA at any test item dose level in either presence or absence of S9-mix.
In the second experiment (pre-incubation method), the test item caused a stronger toxic response with visible reductions in the bacterial lawns noted to all of the tester strains. In the absence of S9-mix, toxicity was initially observed from 500 µg/plate (TA100, TA1537 and TA1535) and 1500 µg/plate (TA98 and WP2uvrA). In the presence of S9-mix, toxicity was initially observed from 500 µg/plate (TA1535), 1500 µg/plate (TA100, TA98 and TA1537) and at 5000 µg/plate (WP2uvrA).
- 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 1 (plate incorporation method) and Experiment 2 (pre incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6
OTHERS:
- 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 in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
Any other information on results incl. tables
Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
Experiment 1 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
81 |
|
23 |
|
27 |
|
29 |
|
13 |
|
81 |
(85) |
19 |
(18) |
14 |
(19) |
27 |
(23) |
14 |
(14) |
92 |
|
13 |
|
15 |
|
14 |
|
14 |
|
Experiment 2 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
79 |
|
18 |
|
22 |
|
27 |
|
8 |
|
61 |
(71) |
21 |
(18) |
19 |
(22) |
26 |
(25) |
9 |
(10) |
73 |
|
16 |
|
24 |
|
21 |
|
12 |
|
Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 08 August 2017 |
To: 11 August 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 (DMSO) |
88 73 79 |
(80) 7.5# |
23 22 21 |
(22) 1.0 |
17 19 33 |
(23) 8.7 |
22 22 21 |
(22) 0.6 |
16 15 13 |
(15) 1.5 |
||
1.5 µg |
66 67 66 |
(66) 0.6 |
19 22 17 |
(19) 2.5 |
19 13 20 |
(17) 3.8 |
20 21 21 |
(21) 0.6 |
6 9 4 |
(6) 2.5 |
||
5 µg |
62 84 81 |
(76) 11.9 |
16 21 23 |
(20) 3.6 |
11 18 14 |
(14) 3.5 |
22 20 20 |
(21) 1.2 |
5 10 11 |
(9) 3.2 |
||
15 µg |
72 65 89 |
(75) 12.3 |
19 12 18 |
(16) 3.8 |
15 21 12 |
(16) 4.6 |
21 24 22 |
(22) 1.5 |
11 11 8 |
(10) 1.7 |
||
50 µg |
75 72 99 |
(82) 14.8 |
16 21 25 |
(21) 4.5 |
19 17 16 |
(17) 1.5 |
21 27 23 |
(24) 3.1 |
13 13 18 |
(15) 2.9 |
||
150 µg |
81 85 95 |
(87) 7.2 |
15 23 21 |
(20) 4.2 |
24 18 23 |
(22) 3.2 |
19 23 22 |
(21) 2.1 |
21 12 12 |
(15) 5.2 |
||
500 µg |
66 78 89 |
(78) 11.5 |
24 15 16 |
(18) 4.9 |
17 20 24 |
(20) 3.5 |
22 18 26 |
(22) 4.0 |
14 10 13 |
(12) 2.1 |
||
1500 µg |
68 SF 46 SF 56 SF |
(57) 11.0 |
13 F 14 F 13 F |
(13) 0.6 |
13 F 14 F 15 F |
(14) 1.0 |
26 F 20 F 16 F |
(21) 5.0 |
3 SF 4 SF 5 SF |
(4) 1.0 |
||
5000 µg |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 VF 0 VF 0 VF |
(0) 0.0 |
14 F 12 F 17 F |
(14) 2.5 |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 VF 0 VF 0 VF |
(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 |
||||||||
702 784 750 |
(745) 41.2 |
544 659 619 |
(607) 58.4 |
618 497 521 |
(545) 64.1 |
198 187 197 |
(194) 6.1 |
274 211 274 |
(253) 36.4 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
F: Test item film
#: Standard deviation
Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 08 August 2017 |
To: 11 August 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 (DMSO) |
88 73 90 |
(84) 9.3# |
18 28 13 |
(20) 7.6 |
27 19 23 |
(23) 4.0 |
23 26 23 |
(24) 1.7 |
8 16 15 |
(13) 4.4 |
||
1.5 µg |
66 80 78 |
(75) 7.6 |
24 17 15 |
(19) 4.7 |
24 23 21 |
(23) 1.5 |
30 26 21 |
(26) 4.5 |
12 10 11 |
(11) 1.0 |
||
5 µg |
103 96 78 |
(92) 12.9 |
20 25 15 |
(20) 5.0 |
20 23 18 |
(20) 2.5 |
16 24 22 |
(21) 4.2 |
9 7 17 |
(11) 5.3 |
||
15 µg |
70 66 93 |
(76) 14.6 |
11 12 13 |
(12) 1.0 |
21 17 30 |
(23) 6.7 |
25 23 34 |
(27) 5.9 |
22 15 9 |
(15) 6.5 |
||
50 µg |
73 62 73 |
(69) 6.4 |
23 12 11 |
(15) 6.7 |
25 23 28 |
(25) 2.5 |
25 23 24 |
(24) 1.0 |
10 11 19 |
(13) 4.9 |
||
150 µg |
78 82 81 |
(80) 2.1 |
14 20 16 |
(17) 3.1 |
22 23 26 |
(24) 2.1 |
22 24 24 |
(23) 1.2 |
17 15 4 |
(12) 7.0 |
||
500 µg |
74 97 82 |
(84) 11.7 |
18 11 18 |
(16) 4.0 |
15 32 29 |
(25) 9.1 |
24 26 25 |
(25) 1.0 |
23 6 5 |
(11) 10.1 |
||
1500 µg |
67 F 85 F 91 F |
(81) 12.5 |
14 F 15 F 15 F |
(15) 0.6 |
14 F 7 F 20 F |
(14) 6.5 |
24 F 23 F 24 F |
(24) 0.6 |
4 F 5 F 5 F |
(5) 0.6 |
||
5000 µg |
17 SF 18 SF 21 SF |
(19) 2.1 |
5 SF 8 SF 6 SF |
(6) 1.5 |
19 F 11 F 19 F |
(16) 4.6 |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 VF 0 VF 0 VF |
(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 |
||||||||
1266 1058 997 |
(1107) 141.0 |
240 315 265 |
(273) 38.2 |
85 97 105 |
(96) 10.1 |
184 210 190 |
(195) 13.6 |
511 398 468 |
(459) 57.0 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
F: Test item film
#: Standard deviation
Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 22 August 2017 |
To: 25 August 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 (DMSO) |
87 76 69 |
(77) 9.1# |
21 11 8 |
(13) 6.8 |
22 17 29 |
(23) 6.0 |
13 12 19 |
(15) 3.8 |
17 15 7 |
(13) 5.3 |
||
1.5 µg |
74 69 76 |
(73) 3.6 |
26 22 24 |
(24) 2.0 |
16 13 21 |
(17) 4.0 |
9 13 14 |
(12) 2.6 |
5 7 10 |
(7) 2.5 |
||
5 µg |
77 70 71 |
(73) 3.8 |
27 22 11 |
(20) 8.2 |
20 17 24 |
(20) 3.5 |
14 18 24 |
(19) 5.0 |
8 12 14 |
(11) 3.1 |
||
15 µg |
69 61 74 |
(68) 6.6 |
22 18 13 |
(18) 4.5 |
22 26 16 |
(21) 5.0 |
16 14 24 |
(18) 5.3 |
15 11 16 |
(14) 2.6 |
||
50 µg |
95 97 67 |
(86) 16.8 |
28 19 18 |
(22) 5.5 |
14 12 15 |
(14) 1.5 |
10 24 17 |
(17) 7.0 |
8 12 6 |
(9) 3.1 |
||
150 µg |
71 67 85 |
(74) 9.5 |
17 17 15 |
(16) 1.2 |
20 16 21 |
(19) 2.6 |
14 22 12 |
(16) 5.3 |
6 9 4 |
(6) 2.5 |
||
500 µg |
23 S 51 S 30 S |
(35) 14.6 |
11 S 9 S 10 S |
(10) 1.0 |
16 24 14 |
(18) 5.3 |
17 10 16 |
(14) 3.8 |
4 S 2 S 2 S |
(3) 1.2 |
||
1500 µg |
23 SF 18 SF 26 SF |
(22) 4.0 |
0 VF 0 VF 0 VF |
(0) 0.0 |
20 SF 17 SF 19 SF |
(19) 1.5 |
14 SF 17 SF 8 SF |
(13) 4.6 |
0 VF 0 VF 0 VF |
(0) 0.0 |
||
5000 µg |
0 TF 0 TF 0 TF |
(0) 0.0 |
0 TF 0 TF 0 TF |
(0) 0.0 |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 TF 0 TF 0 TF |
(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 |
||||||||
766 812 900 |
(826) 68.1 |
546 515 490 |
(517) 28.1 |
593 804 697 |
(698) 105.5 |
239 240 249 |
(243) 5.5 |
185 175 119 |
(160) 35.6 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
F: Test item film
T: Toxic, no bacterial background lawn
#: Standard deviation
Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 22 August 2017 |
To: 25 August 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 (DMSO) |
104 76 82 |
(87) 14.7# |
18 10 27 |
(18) 8.5 |
19 37 23 |
(26) 9.5 |
16 25 19 |
(20) 4.6 |
10 10 10 |
(10) 0.0 |
||
1.5 µg |
79 86 88 |
(84) 4.7 |
16 21 17 |
(18) 2.6 |
30 25 25 |
(27) 2.9 |
18 23 22 |
(21) 2.6 |
15 12 12 |
(13) 1.7 |
||
5 µg |
63 65 74 |
(67) 5.9 |
21 15 21 |
(19) 3.5 |
34 21 20 |
(25) 7.8 |
31 22 19 |
(24) 6.2 |
12 14 8 |
(11) 3.1 |
||
15 µg |
77 67 66 |
(70) 6.1 |
18 15 15 |
(16) 1.7 |
23 17 21 |
(20) 3.1 |
26 26 22 |
(25) 2.3 |
10 15 10 |
(12) 2.9 |
||
50 µg |
65 71 115 |
(84) 27.3 |
14 22 25 |
(20) 5.7 |
24 26 28 |
(26) 2.0 |
28 30 20 |
(26) 5.3 |
6 7 9 |
(7) 1.5 |
||
150 µg |
74 78 62 |
(71) 8.3 |
13 7 17 |
(12) 5.0 |
31 27 17 |
(25) 7.2 |
28 16 26 |
(23) 6.4 |
10 9 11 |
(10) 1.0 |
||
500 µg |
38 42 70 |
(50) 17.4 |
18 S 23 S 19 S |
(20) 2.6 |
33 30 21 |
(28) 6.2 |
21 31 21 |
(24) 5.8 |
7 8 13 |
(9) 3.2 |
||
1500 µg |
31 SF 32 SF 27 SF |
(30) 2.6 |
0 VF 0 VF 0 VF |
(0) 0.0 |
26 F 15 F 25 F |
(22) 6.1 |
12 SF 10 SF 24 SF |
(15) 7.6 |
3 SF 2 SF 4 SF |
(3) 1.0 |
||
5000 µg |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 TF 0 TF 0 TF |
(0) 0.0 |
8 SF 11 SF 10 SF |
(10) 1.5 |
0 VF 0 VF 0 VF |
(0) 0.0 |
0 TF 0 TF 0 TF |
(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 |
||||||||
1029 900 994 |
(974) 66.7 |
208 188 203 |
(200) 10.4 |
217 168 213 |
(199) 27.2 |
110 132 134 |
(125) 13.3 |
337 371 349 |
(352) 17.2 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
F: Test item film
T: Toxic, no bacterial background lawn
#: Standard deviation
Table 7.6.1/6: History Profile of Vehicle and Positive Control Values
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values† |
274 |
278 |
504 |
285 |
26 |
13 |
461 |
229 |
526 |
299 |
506 |
282 |
42 |
51 |
39 |
49 |
|||||||||||||||||
Min |
60 |
61 |
7 |
7 |
222 |
278 |
10 |
12 |
11 |
10 |
4 |
6 |
87 |
98 |
89 |
93 |
|||||||||||||||||
Max |
166 |
175 |
31 |
29 |
376 |
388 |
58 |
43 |
45 |
46 |
27 |
27 |
237 |
254 |
174 |
177 |
|||||||||||||||||
Mean |
91 |
95 |
16 |
14 |
286 |
333 |
24 |
27 |
21 |
24 |
12 |
13 |
156 |
164 |
123 |
137 |
|||||||||||||||||
SD |
19.3 |
19.1 |
4.5 |
4.0 |
48.7 |
37.6 |
5.6 |
5.9 |
6.2 |
6.1 |
3.8 |
3.4 |
42.2 |
35.6 |
23.1 |
21.2 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2015 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
276 |
280 |
252 |
264 |
13 |
13 |
231 |
227 |
262 |
276 |
253 |
261 |
20 |
35 |
20 |
35 |
|
||||||||||||||||
Min |
222 |
250 |
79 |
118 |
953 |
673 |
116 |
103 |
100 |
78 |
164 |
97 |
430 |
494 |
745 |
325 |
|
||||||||||||||||
Max |
2266 |
2402 |
2779 |
457 |
3140 |
1655 |
2769 |
550 |
502 |
705 |
2318 |
823 |
1696 |
2264 |
3662 |
1174 |
|
||||||||||||||||
Mean |
614 |
927 |
472 |
246 |
2303 |
1093 |
792 |
266 |
222 |
218 |
911 |
336 |
761 |
1461 |
2257 |
569 |
|
||||||||||||||||
SD |
260.6 |
452.5 |
434.8 |
55.7 |
815.2 |
376.5 |
342.1 |
97.7 |
70.2 |
107.6 |
412.4 |
135.7 |
350.0 |
382.0 |
790.7 |
220.3 |
|
||||||||||||||||
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values |
399 |
401 |
758 |
393 |
60 |
30 |
690 |
345 |
788 |
415 |
762 |
398 |
32 |
32 |
16 |
24 |
|||||||||||||||||
Min |
63 |
66 |
8 |
8 |
216 |
221 |
10 |
13 |
8 |
12 |
3 |
4 |
97 |
104 |
78 |
52 |
|||||||||||||||||
Max |
154 |
156 |
34 |
39 |
340 |
375 |
53 |
53 |
49 |
51 |
24 |
23 |
268 |
243 |
148 |
166 |
|||||||||||||||||
Mean |
90 |
93 |
15 |
15 |
268 |
310 |
22 |
27 |
21 |
25 |
12 |
13 |
161 |
159 |
118 |
110 |
|||||||||||||||||
SD |
14.5 |
14.3 |
4.5 |
5.2 |
26.4 |
31.1 |
5.8 |
6.3 |
4.8 |
5.7 |
3.5 |
3.5 |
39.2 |
32.3 |
17.0 |
29.3 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2016 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
409 |
406 |
381 |
386 |
30 |
28 |
341 |
335 |
388 |
385 |
379 |
381 |
14 |
24 |
8 |
16 |
|
||||||||||||||||
Min |
221 |
284 |
84 |
92 |
897 |
629 |
107 |
102 |
100 |
96 |
95 |
101 |
445 |
574 |
1674 |
372 |
|
||||||||||||||||
Max |
2222 |
2863 |
2994 |
879 |
2326 |
2140 |
1611 |
637 |
449 |
4357 |
1413 |
639 |
1117 |
1855 |
2823 |
945 |
|
||||||||||||||||
Mean |
724 |
1264 |
854 |
240 |
1633 |
950 |
718 |
240 |
186 |
188 |
406 |
290 |
743 |
1271 |
2379 |
535 |
|
||||||||||||||||
SD |
320.4 |
562.9 |
664.9 |
62.1 |
564.5 |
382.7 |
338.6 |
98.2 |
49.8 |
230.8 |
227.0 |
92.7 |
214.6 |
326.5 |
426.2 |
143.3 |
|
||||||||||||||||
Applicant's summary and conclusion
- Conclusions:
- Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
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.
In the first experiment (plate incorporation), the test item caused a visible reduction in the growth of the Salmonella bacterial strains initially at 1500 and at 5000 µg/plate in the absence and presence of metabolic activation (S9), respectively.
In the second experiment, the test item caused a visible reduction in the growth of the bacterial background lawns initially at 500 µg/plate in both the absence and presence of metabolic activation (S9).
A test item induced film (light yellow in colour) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
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 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
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