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EC number: 289-860-8 | CAS number: 90028-67-4 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Evernia furfuracea, 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 - 28 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:
- Pin-2(3)-ene
- EC Number:
- 201-291-9
- EC Name:
- Pin-2(3)-ene
- Cas Number:
- 80-56-8
- Molecular formula:
- C10H16
- IUPAC Name:
- 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene
- Test material form:
- other: black-brown, sticky high viscous resin
- Details on test material:
- Name TREEMOSS ABSOLUTE (as reported in the report)
Batch no. 0416/1
Appearance Black-brown, sticky high viscous resin
Composition 100% TREEMOSS ABSOLUTE
CAS No. 90028-67-4
EINECS-No. 289-860-8
Production date 16. Apr. 2016
Expiry date 15.Apr. 2018
Storage Room temp. (20+-5°C), Keep away from light and humidity, keep under inert gas
Constituent 1
Constituent 2
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0416/1
- Expiration date of the lot/batch: 15 April 2018
- Purity test date: April 2016
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, on the day of each experiment, approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at approximately 40°C. 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 and 1500 µg/plate, with and without S9-mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: In solubility checks performed in house, the test item was noted as insoluble in sterile distilled water at 50 mg/mL but fully soluble in dimethyl sulphoxide at the same concentration and in acetone at 100 mg/mL. Dimethyl sulphoxide was 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). Several 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 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 1.5 to 1500 µg/plate. Seven 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: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
MUTAGENICITY
- The vehicle (dimethyl sulphoxide (DMSO)) 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 bacterial background lawn in all of the tester strains both with and without metabolic activation (S9-mix) at and above 1500 µg/plate.
In the second experiment (pre-incubation), the test item caused a visible reduction in all of the bacterial lawns in the absence of S9-mix, initially from 500 µg/plate (TA100, TA1535, TA98 and TA1537) and at 1500 µg/plate (WP2uvrA). In the presence of S9-mix toxicity was noted to all of the tester strains at 1500 µg/plate.
- 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. A single count for TA1535 (untreated control in the second mutation test) was just above the maximum historical control range. This count was still considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9 mix.
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 |
|||||
118 |
|
16 |
|
17 |
|
21 |
|
17 |
|
91 |
(99) |
16 |
(20) |
15 |
(17) |
16 |
(21) |
9 |
(11) |
89 |
|
27 |
|
19 |
|
25 |
|
8 |
|
Experiment 2 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
91 |
|
32 |
|
25 |
|
20 |
|
17 |
|
94 |
(91) |
44 |
(38) |
39 |
(33) |
18 |
(22) |
6 |
(13) |
89 |
|
37 |
|
36 |
|
28 |
|
15 |
|
Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 09 August 2017 |
To: 12 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) |
88 107 118 |
(104) 15.2# |
18 17 14 |
(16) 2.1 |
19 13 17 |
(16) 3.1 |
22 17 23 |
(21) 3.2 |
10 13 18 |
(14) 4.0 |
||
1.5 µg |
94 113 97 |
(101) 10.2 |
15 19 17 |
(17) 2.0 |
16 15 19 |
(17) 2.1 |
23 13 28 |
(21) 7.6 |
6 12 10 |
(9) 3.1 |
||
5 µg |
113 103 114 |
(110) 6.1 |
11 16 15 |
(14) 2.6 |
25 17 15 |
(19) 5.3 |
22 22 22 |
(22) 0.0 |
12 11 10 |
(11) 1.0 |
||
15 µg |
86 105 116 |
(102) 15.2 |
14 14 17 |
(15) 1.7 |
14 16 14 |
(15) 1.2 |
23 19 20 |
(21) 2.1 |
10 11 10 |
(10) 0.6 |
||
50 µg |
119 81 114 |
(105) 20.6 |
19 11 21 |
(17) 5.3 |
18 17 11 |
(15) 3.8 |
23 24 26 |
(24) 1.5 |
15 16 14 |
(15) 1.0 |
||
150 µg |
117 110 111 |
(113) 3.8 |
17 16 16 |
(16) 0.6 |
15 19 19 |
(18) 2.3 |
16 24 20 |
(20) 4.0 |
13 14 13 |
(13) 0.6 |
||
500 µg |
119 90 67 |
(92) 26.1 |
14 15 11 |
(13) 2.1 |
19 16 17 |
(17) 1.5 |
21 22 20 |
(21) 1.0 |
13 11 12 |
(12) 1.0 |
||
1500 µg |
77 S 88 S 70 S |
(78) 9.1 |
19 S 8 S 11 S |
(13) 5.7 |
19 S 19 S 15 S |
(18) 2.3 |
16 S 18 S 15 S |
(16) 1.5 |
13 S 15 S 11 S |
(13) 2.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
7 S 8 S 7 S |
(7) 0.6 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(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 |
||||||||
648 801 864 |
(771) 111.1 |
509 673 750 |
(644) 123.1 |
837 854 856 |
(849) 10.4 |
258 230 227 |
(238) 17.1 |
309 622 640 |
(524) 186.1 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
T: Toxic background lawn
S: Sparse bacterial background lawn
#: Standard deviation
Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 09 August 2017 |
To: 12 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) |
100 107 95 |
(101) 6.0# |
13 21 15 |
(16) 4.2 |
22 27 29 |
(26) 3.6 |
27 21 23 |
(24) 3.1 |
14 15 22 |
(17) 4.4 |
||
1.5 µg |
107 115 95 |
(106) 10.1 |
13 18 22 |
(18) 4.5 |
14 33 22 |
(23) 9.5 |
23 22 27 |
(24) 2.6 |
12 12 11 |
(12) 0.6 |
||
5 µg |
102 99 95 |
(99) 3.5 |
14 18 21 |
(18) 3.5 |
26 31 14 |
(24) 8.7 |
28 25 30 |
(28) 2.5 |
10 7 13 |
(10) 3.0 |
||
15 µg |
104 97 122 |
(108) 12.9 |
17 18 20 |
(18) 1.5 |
34 28 25 |
(29) 4.6 |
31 31 26 |
(29) 2.9 |
11 29 17 |
(19) 9.2 |
||
50 µg |
111 103 119 |
(111) 8.0 |
14 19 20 |
(18) 3.2 |
17 38 18 |
(24) 11.8 |
27 29 28 |
(28) 1.0 |
11 18 20 |
(16) 4.7 |
||
150 µg |
76 99 92 |
(89) 11.8 |
20 17 14 |
(17) 3.0 |
26 30 21 |
(26) 4.5 |
28 19 23 |
(23) 4.5 |
7 17 10 |
(11) 5.1 |
||
500 µg |
125 107 82 |
(105) 21.6 |
14 16 15 |
(15) 1.0 |
31 22 18 |
(24) 6.7 |
33 33 28 |
(31) 2.9 |
15 14 10 |
(13) 2.6 |
||
1500 µg |
88 S 82 S 88 S |
(86) 3.5 |
11 S 8 S 7 S |
(9) 2.1 |
22 S 26 S 20 S |
(23) 3.1 |
19 S 27 S 17 S |
(21) 5.3 |
4 S 4 S 6 S |
(5) 1.2 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
3 S 3 S 4 S |
(3) 0.6 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(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 |
||||||||
1012 1062 1227 |
(1100) 112.5 |
267 267 258 |
(264) 5.2 |
127 147 152 |
(142) 13.2 |
197 162 192 |
(184) 18.9 |
424 460 459 |
(448) 20.5 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
T: Toxic background lawn
S: Sparse bacterial background lawn
#: Standard deviation
Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 24 August 2017 |
To: 27 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) |
77 69 64 |
(70) 6.6# |
23 25 38 |
(29) 8.1 |
27 30 25 |
(27) 2.5 |
22 20 21 |
(21) 1.0 |
5 16 6 |
(9) 6.1 |
||
1.5 µg |
79 87 66 |
(77) 10.6 |
18 34 24 |
(25) 8.1 |
30 26 17 |
(24) 6.7 |
20 15 8 |
(14) 6.0 |
12 10 10 |
(11) 1.2 |
||
5 µg |
77 62 66 |
(68) 7.8 |
36 24 29 |
(30) 6.0 |
31 25 23 |
(26) 4.2 |
21 23 13 |
(19) 5.3 |
6 6 10 |
(7) 2.3 |
||
15 µg |
73 76 60 |
(70) 8.5 |
28 39 27 |
(31) 6.7 |
27 41 32 |
(33) 7.1 |
20 21 21 |
(21) 0.6 |
7 7 9 |
(8) 1.2 |
||
50 µg |
65 70 69 |
(68) 2.6 |
19 22 27 |
(23) 4.0 |
41 35 32 |
(36) 4.6 |
15 24 13 |
(17) 5.9 |
9 12 11 |
(11) 1.5 |
||
150 µg |
64 58 66 |
(63) 4.2 |
20 39 22 |
(27) 10.4 |
23 18 30 |
(24) 6.0 |
15 27 21 |
(21) 6.0 |
8 18 8 |
(11) 5.8 |
||
500 µg |
62 S 37 S 37 S |
(45) 14.4 |
19 S 28 S 30 S |
(26) 5.9 |
20 25 16 |
(20) 4.5 |
10 S 15 S 20 S |
(15) 5.0 |
5 S 4 S 7 S |
(5) 1.5 |
||
1500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(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 |
||||||||
1187 1012 969 |
(1056) 115.5 |
612 732 691 |
(678) 61.0 |
1012 1014 948 |
(991) 37.5 |
273 302 310 |
(295) 19.5 |
322 308 282 |
(304) 20.3 |
|||
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
#: Standard deviation
Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 24 August 2017 |
To: 27 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) |
75 73 72 |
(73) 1.5# |
20 28 27 |
(25) 4.4 |
36 39 37 |
(37) 1.5 |
20 33 33 |
(29) 7.5 |
11 10 13 |
(11) 1.5 |
||
1.5 µg |
60 58 62 |
(60) 2.0 |
25 26 16 |
(22) 5.5 |
40 44 41 |
(42) 2.1 |
42 26 35 |
(34) 8.0 |
12 10 12 |
(11) 1.2 |
||
5 µg |
61 65 57 |
(61) 4.0 |
32 33 25 |
(30) 4.4 |
35 36 37 |
(36) 1.0 |
15 34 24 |
(24) 9.5 |
15 13 12 |
(13) 1.5 |
||
15 µg |
52 77 85 |
(71) 17.2 |
25 27 28 |
(27) 1.5 |
32 33 40 |
(35) 4.4 |
36 24 26 |
(29) 6.4 |
14 8 19 |
(14) 5.5 |
||
50 µg |
69 61 53 |
(61) 8.0 |
26 26 23 |
(25) 1.7 |
36 32 36 |
(35) 2.3 |
26 30 26 |
(27) 2.3 |
6 13 16 |
(12) 5.1 |
||
150 µg |
55 59 61 |
(58) 3.1 |
23 26 24 |
(24) 1.5 |
21 40 39 |
(33) 10.7 |
16 22 20 |
(19) 3.1 |
12 15 11 |
(13) 2.1 |
||
500 µg |
39 50 40 |
(43) 6.1 |
24 23 24 |
(24) 0.6 |
35 32 28 |
(32) 3.5 |
19 12 27 |
(19) 7.5 |
7 8 7 |
(7) 0.6 |
||
1500 µg |
35 S 45 S 39 S |
(40) 5.0 |
0 V 0 V 0 V |
(0) 0.0 |
18 S 22 S 23 S |
(21) 2.6 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(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 |
||||||||
1296 1278 1223 |
(1266) 38.0 |
244 252 278 |
(258) 17.8 |
312 304 317 |
(311) 6.6 |
152 179 151 |
(161) 15.9 |
522 573 471 |
(522) 51.0 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
S: Sparse bacterial background lawn
V: Very weak 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 and 1500 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 bacterial background lawn in all of the tester strains both with and without metabolic activation (S9-mix) at and above 1500 µg/plate.
In the second experiment (pre-incubation), the test item caused a visible reduction in all of the bacterial lawns in the absence of S9-mix, initially from 500 µg/plate (TA100, TA1535, TA98 and TA1537) and at 1500 µg/plate (WP2uvrA). In the presence of S9-mix toxicity was noted to all of the tester strains at 1500 µg/plate.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test 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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