Registration Dossier
Registration Dossier
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EC number: 282-029-0 | CAS number: 84082-82-6 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Salix alba, Salicaceae.
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:
- 12.02.2019 - 28.06.2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- version from 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- α-hydroxy-o-tolyl β-D-glucopyranoside
- EC Number:
- 205-331-6
- EC Name:
- α-hydroxy-o-tolyl β-D-glucopyranoside
- Cas Number:
- 138-52-3
- Molecular formula:
- C13H18O7
- IUPAC Name:
- (2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[2-(hydroxymethyl)phenoxy]oxane-3,4,5-triol
- Test material form:
- solid: particulate/powder
- Details on test material:
- Name: White willow bark extract
CAS number: 84082-82-6
Specification:
Purity: 100% (UVCB substance)
Appearance: Brown powder
Odour: Characteristic
Sieve analysis: 100% pass 80 mesh
Loss on drying: 5.0 % max
Residue on ignition: 5.0 % max
Bulk density: 40 – 55 g/100ml
Extract solvent: Alcohol & water
Heavy metal: 10 ppm max
Lead (Pb): 2 ppm max
Arsenic (As): 2 ppm max
Cadmium (Cd): 2 ppm max
Mercury (Hg): 2 ppm max
Residual solvents: Complies with Eur.Pharm.
Microbiological:
===============
Total Plate Count: 1000 cfu/g max
Yeast & Mould: 100 cfu/g max
E. coli: Negative
Salmonella: Negative
Constituent 1
Method
- Target gene:
- trp/his
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- plate incorporation method: 5000, 1581, 500, 158.1, 50, 15.81 and 0 μg/plate. 5000 µg/plate is the highest recommended dose according to the guideline.
pre-incubation method: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0 μg/plate. Doses higher than 500 showed excessive cytotoxicity. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF), Acetone, Ethanol and n-Hexane. The highest solubility was achieved using DMSO, at a concentration of 50 mg/mL.
- Justification for percentage of solvent in the final culture medium: highest soluble concentration
Controls
- Untreated negative controls:
- yes
- Remarks:
- With and without S9
- Negative solvent / vehicle controls:
- yes
- Remarks:
- With and without S9
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With and without S9
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylene-diamine (NPD) 2-Aminoanthracene (2-AA)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1 plate-incorporation, 1 pre-incubation
METHOD OF TREATMENT/ EXPOSURE:
plate incorporation:
Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 µL
vehicle or test item formulation (or reference controls) 100 (50)* µL
overnight culture of test strain 100 µL
phosphate buffer (pH 7.4) or S9 mix 500 µL
*Note: Treatment volume was 100 µL for test item formulations and its solvent; treatment volume was 50 µL for positive control substances and their solvents.
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
preincubation :
Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 5.3.6.) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
- Rationale for test conditions:
- Guideline
- Evaluation criteria:
- The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor (mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test item and for the controls.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analyzable concentrations are presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a Negative Response:
The test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation - Statistics:
- No statistical testing was performed, as the results were unambigious according to the evaluation criteria.
Results and discussion
Test results
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- for the pre-incubation assay, concentrations above 500 µg/plate showed excessive toxicity. For the plate incorporation method, the highest recommended test item concentration according to the guideline was used.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Summary Table of the plate incorporation assay (Assay 1)
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
16.3 |
20.7 |
98.7 |
100.0 |
13.3 |
12.7 |
9.3 |
7.7 |
47.0 |
49.3 |
|
MF |
0.96 |
1.02 |
1.05 |
1.02 |
1.21 |
1.23 |
1.00 |
0.82 |
1.02 |
0.98 |
||
DMSO control 50µL |
Mean |
17.7 |
20.3 |
-- |
97.7 |
-- |
13.0 |
6.7 |
8.0 |
-- |
48.7 |
|
MF |
1.04 |
1.00 |
-- |
1.00 |
-- |
1.26 |
0.71 |
0.86 |
-- |
0.97 |
||
Distilled water control |
Mean |
-- |
-- |
97.0 |
-- |
11.7 |
-- |
-- |
-- |
47.3 |
-- |
|
MF |
-- |
-- |
1.03 |
-- |
1.06 |
-- |
-- |
-- |
1.03 |
-- |
||
DMSO 100µL control |
Mean |
17.0 |
20.3 |
94.3 |
98.0 |
11.0 |
10.3 |
9.3 |
9.3 |
46.0 |
50.3 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
19.3 |
22.3 |
83.7 |
101.7 |
17.0 |
14.7 |
8.0 |
8.3 |
44.0 |
53.3 |
|
MF |
1.14 |
1.10 |
0.89 |
1.04 |
1.55 |
1.42 |
0.86 |
0.89 |
0.96 |
1.06 |
||
1581 |
Mean |
18.7 |
24.3 |
91.7 |
105.3 |
11.3 |
14.0 |
6.7 |
9.3 |
46.0 |
50.7 |
|
MF |
1.10 |
1.20 |
0.97 |
1.07 |
1.03 |
1.35 |
0.71 |
1.00 |
1.00 |
1.01 |
||
500 |
Mean |
16.3 |
23.7 |
97.3 |
104.3 |
11.0 |
12.3 |
7.3 |
8.0 |
46.3 |
49.7 |
|
MF |
0.96 |
1.16 |
1.03 |
1.06 |
1.00 |
1.19 |
0.79 |
0.86 |
1.01 |
0.99 |
||
158.1 |
Mean |
17.3 |
23.3 |
93.0 |
100.3 |
11.3 |
9.7 |
7.3 |
9.0 |
46.3 |
50.0 |
|
MF |
1.02 |
1.15 |
0.99 |
1.02 |
1.03 |
0.94 |
0.79 |
0.96 |
1.01 |
0.99 |
||
50 |
Mean |
17.0 |
19.0 |
89.7 |
98.7 |
10.7 |
10.7 |
7.3 |
9.0 |
47.3 |
49.0 |
|
MF |
1.00 |
0.93 |
0.95 |
1.01 |
0.97 |
1.03 |
0.79 |
0.96 |
1.03 |
0.97 |
||
15.81 |
Mean |
15.7 |
19.7 |
99.0 |
96.7 |
12.3 |
11.7 |
8.3 |
9.0 |
46.7 |
50.0 |
|
MF |
0.92 |
0.97 |
1.05 |
0.99 |
1.12 |
1.13 |
0.89 |
0.96 |
1.01 |
0.99 |
||
NPD (4mg) |
Mean |
405.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
22.94 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2409.3 |
-- |
2488.0 |
-- |
204.0 |
-- |
302.0 |
-- |
-- |
|
MF |
-- |
118.49 |
-- |
25.47 |
-- |
15.69 |
-- |
37.75 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
249.3 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.12 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1036.0 |
-- |
1129.3 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
10.68 |
-- |
96.80 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
409.3 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
61.40 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1098.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
23.21 |
-- |
||
Summary Table of the pre-incubation assay (Assay 3)
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
|||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||
Untreated control |
Mean |
18.7 |
20.7 |
95.0 |
93.3 |
12.3 |
14.0 |
7.7 |
10.7 |
46.7 |
50.0 |
||
MF |
0.98 |
1.00 |
1.21 |
1.05 |
0.90 |
1.05 |
1.00 |
1.33 |
1.06 |
1.01 |
|||
DMSO control 50µL |
Mean |
17.7 |
20.0 |
-- |
91.0 |
-- |
14.3 |
7.3 |
7.7 |
-- |
49.0 |
||
MF |
0.93 |
0.97 |
-- |
1.02 |
-- |
1.08 |
0.96 |
0.96 |
-- |
0.99 |
|||
Distilled water control |
Mean |
-- |
-- |
86.7 |
-- |
14.0 |
-- |
-- |
-- |
44.7 |
-- |
||
MF |
-- |
-- |
1.11 |
-- |
1.02 |
-- |
-- |
-- |
1.02 |
-- |
|||
DMSO 100µL control |
Mean |
19.0 |
20.7 |
78.3 |
89.0 |
13.7 |
13.3 |
7.7 |
8.0 |
44.0 |
49.3 |
||
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|||
500 |
Mean |
13.3 |
21.7 |
70.7 |
98.3 |
10.7 |
13.0 |
8.3 |
10.3 |
40.0 |
49.3 |
||
MF |
0.70 |
1.05 |
0.90 |
1.10 |
0.78 |
0.98 |
1.09 |
1.29 |
0.91 |
1.00 |
|||
158.1 |
Mean |
15.3 |
21.0 |
83.7 |
97.7 |
11.7 |
11.3 |
5.3 |
10.0 |
41.0 |
49.3 |
||
MF |
0.81 |
1.02 |
1.07 |
1.10 |
0.85 |
0.85 |
0.70 |
1.25 |
0.93 |
1.00 |
|||
50 |
Mean |
20.0 |
21.7 |
88.3 |
91.7 |
11.7 |
12.0 |
9.3 |
8.3 |
42.7 |
49.3 |
||
MF |
1.05 |
1.05 |
1.13 |
1.03 |
0.85 |
0.90 |
1.22 |
1.04 |
0.97 |
1.00 |
|||
15.81 |
Mean |
18.7 |
19.7 |
86.3 |
92.0 |
9.0 |
12.7 |
8.7 |
9.7 |
44.3 |
51.0 |
||
MF |
0.98 |
0.95 |
1.10 |
1.03 |
0.66 |
0.95 |
1.13 |
1.21 |
1.01 |
1.03 |
|||
5 |
Mean |
19.3 |
26.3 |
88.3 |
89.3 |
12.0 |
11.7 |
8.3 |
10.7 |
40.0 |
50.0 |
||
MF |
1.02 |
1.27 |
1.13 |
1.00 |
0.88 |
0.88 |
1.09 |
1.33 |
0.91 |
1.01 |
|||
1.581 |
Mean |
16.7 |
23.3 |
84.3 |
94.0 |
12.3 |
12.3 |
8.3 |
8.0 |
40.3 |
49.7 |
||
MF |
0.88 |
1.13 |
1.08 |
1.06 |
0.90 |
0.93 |
1.09 |
1.00 |
0.92 |
1.01 |
|||
0.5 |
Mean |
16.0 |
23.7 |
89.7 |
92.7 |
12.0 |
10.7 |
8.0 |
11.3 |
40.7 |
48.0 |
||
MF |
0.84 |
1.15 |
1.14 |
1.04 |
0.88 |
0.80 |
1.04 |
1.42 |
0.92 |
0.97 |
|||
NPD (4mg) |
Mean |
406.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
MF |
23.02 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|||
2AA (2mg) |
Mean |
-- |
2430.7 |
-- |
2460.0 |
-- |
219.3 |
-- |
208.0 |
-- |
-- |
||
MF |
-- |
121.53 |
-- |
27.03 |
-- |
15.30 |
-- |
27.13 |
-- |
-- |
|||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
258.0 |
||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.27 |
|||
SAZ (2mg) |
Mean |
-- |
-- |
1065.3 |
-- |
1234.7 |
-- |
-- |
-- |
-- |
-- |
||
MF |
-- |
-- |
12.29 |
-- |
88.19 |
-- |
-- |
-- |
-- |
-- |
|||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
410.7 |
-- |
-- |
-- |
||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
56.00 |
-- |
-- |
-- |
|||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1109.3 |
-- |
||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
24.84 |
-- |
|||
Applicant's summary and conclusion
- Conclusions:
- The test item has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
- Executive summary:
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD TG 471 and in compliance with GLP.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/beta-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-Incubation Method) and an Assay 3 (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/mL. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA98 and TA100 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate and in the Assay 3 were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
In the Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in all tested bacterial strains, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains with and without metabolic activation was repeated in Assay 3 using a modified concentration range. Results of the invalid experiment were not reported; however, all data will be kept and archived in the raw data binder.
No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effects of the test item (reduced / slightly reduced background lawn development) was observed in the Assay 3 in all tested bacterial strains with and without metabolic activation at higher concentrations.
In the Assay 1 and Assay 3, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the bacterial strains under the test conditions used in this study.
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