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EC number: 949-711-6 | CAS number: -
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 June 2020 to
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Saccharomyces cerevisiae cell wall, extracted
- EC Number:
- 949-711-6
- Molecular formula:
- Not applicable (UVCB substance)
- IUPAC Name:
- Saccharomyces cerevisiae cell wall, extracted
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch number of test material: AD19K01750
- Expiration date of the lot/batch: 27 July 2021
Method
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:
- Type and composition of metabolic activation system:
- source of S9 : Rat (Wistar) Liver Homogenate
- method of preparation of S9 mix : Rats treated with phenobarbital (PB) and -naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. On day 4, washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained.
- Volume of S9 mix in the final culture medium: 500 µL - Test concentrations with justification for top dose:
- 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate, for all strains and with/without activation.
Precipitation and cytotoxicity observed down to 158.1 µg/plate with and without activation.
Cytox - Vehicle / solvent:
- - Vehicle used: Distilled water
- Justification for choice of vehicle: Suitable homogenous light beige suspension obtained with distilled water, not with neither DMSO nor DMF.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-1,2-phenylene-diamine (NPD) Without S9 - Salmonella TA98 Methyl-methanesulfonate (MMS) Without S9 - E.coli WP2 uvrA 2-aminoanthracene (2AA) With S9 - All Salmonella strains and E.coli WP2 uvrA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Ranging from 1.39 x10-9 CFU/mL to 3.2x10-9 CFU/mL.
- Test substance added in medium:
* Assay 1: Direct incorporation then in agar plate.
* Assay 2 (all strains except S. TA1535)/3 (S. TA1535 only): Preincupation then in agar plate.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: Assay 2 (all strains except S. TA1535)/3 (S. TA1535 only): 20 minutes at 37°C.
- Exposure duration/duration of treatment: 48h at 37°C
- Harvest time after the end of treatment : at the end of the treatment period.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduced/slight reduced background lawn development - Evaluation criteria:
- A test item is 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.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to cytotoxicity/precipitating concentrations (500 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to cytotoxicity/precipitating concentrations (500 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to cytotoxicity/precipitating concentrations (500 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to cytotoxicity/precipitating concentrations (500 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: tested up to cytotoxicity/precipitating concentrations (500 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
Any other information on results incl. tables
Table1:Summary Table of the Assay 1
Concentrations
|
Mean values ofrevertants/ 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 |
18.0 |
20.3 |
86.7 |
95.3 |
16.3 |
16.0 |
12.0 |
12.0 |
44.0 |
44.7 |
|
MF |
1.04 |
0.87 |
1.10 |
0.91 |
1.02 |
0.94 |
1.06 |
1.03 |
1.06 |
1.01 |
||
DMSO control |
Mean |
18.3 |
21.3 |
-- |
94.7 |
-- |
15.7 |
11.3 |
11.3 |
-- |
46.0 |
|
MF |
1.06 |
0.91 |
-- |
0.90 |
-- |
0.92 |
1.00 |
0.97 |
-- |
1.04 |
||
Distilled water control |
Mean |
17.3 |
23.3 |
79.0 |
105.3 |
16.0 |
17.0 |
11.3 |
11.7 |
41.7 |
44.3 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
500 R/P |
Mean |
16.0 |
20.7 |
89.3 |
95.7 |
16.3 |
16.3 |
11.3 |
11.7 |
45.3 |
43.0 |
|
MF |
0.92 |
0.89 |
1.13 |
0.91 |
1.02 |
0.96 |
1.00 |
1.00 |
1.09 |
0.97 |
||
158.1 SR/P |
Mean |
15.7 |
21.0 |
100.0 |
111.7 |
15.3 |
16.0 |
10.3 |
10.7 |
45.7 |
46.7 |
|
MF |
0.90 |
0.90 |
1.27 |
1.06 |
0.96 |
0.94 |
0.91 |
0.91 |
1.10 |
1.05 |
||
50 |
Mean |
18.3 |
19.7 |
88.3 |
114.3 |
17.0 |
15.7 |
11.3 |
10.7 |
44.0 |
42.3 |
|
MF |
1.06 |
0.84 |
1.12 |
1.09 |
1.06 |
0.92 |
1.00 |
0.91 |
1.06 |
0.95 |
||
15.81 |
Mean |
17.3 |
21.0 |
89.3 |
115.0 |
16.7 |
16.7 |
8.7 |
10.0 |
43.7 |
44.7 |
|
MF |
1.00 |
0.90 |
1.13 |
1.09 |
1.04 |
0.98 |
0.76 |
0.86 |
1.05 |
1.01 |
||
5 |
Mean |
14.3 |
21.0 |
98.0 |
107.7 |
16.3 |
15.3 |
9.3 |
11.0 |
41.7 |
42.7 |
|
MF |
0.83 |
0.90 |
1.24 |
1.02 |
1.02 |
0.90 |
0.82 |
0.94 |
1.00 |
0.96 |
||
1.581 |
Mean |
14.7 |
20.0 |
101.0 |
112.0 |
16.3 |
15.7 |
10.7 |
11.3 |
41.0 |
45.7 |
|
MF |
0.85 |
0.86 |
1.28 |
1.06 |
1.02 |
0.92 |
0.94 |
0.97 |
0.98 |
1.03 |
||
0.5 |
Mean |
17.3 |
19.0 |
102.0 |
105.7 |
16.7 |
16.0 |
10.7 |
10.7 |
43.3 |
45.7 |
|
MF |
1.00 |
0.81 |
1.29 |
1.00 |
1.04 |
0.94 |
0.94 |
0.91 |
1.04 |
1.03 |
||
0.1581 |
Mean |
17.7 |
19.0 |
90.3 |
101.7 |
16.3 |
15.0 |
9.7 |
11.0 |
43.3 |
46.0 |
|
MF |
1.02 |
0.81 |
1.14 |
0.97 |
1.02 |
0.88 |
0.85 |
0.94 |
1.04 |
1.04 |
||
NPD (4mg) |
Mean |
417.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
22.76 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2469.3 |
-- |
2468.0 |
-- |
219.0 |
-- |
210.3 |
-- |
-- |
|
MF |
-- |
115.75 |
-- |
26.07 |
-- |
13.98 |
-- |
18.56 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
238.0 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.17 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1060.0 |
-- |
1217.3 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
13.42 |
-- |
76.08 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
412.0 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
36.35 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1085.3 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
26.05 |
-- |
||
Table2:Summary Table of the Assay 2 and Assay 3
Concentrations
|
Mean values ofrevertants/ 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 |
16.0 |
19.0 |
91.0 |
103.3 |
17.0 |
16.3 |
8.0 |
11.0 |
44.3 |
48.3 |
|
|
MF |
0.91 |
0.93 |
1.09 |
0.97 |
1.00 |
1.09 |
1.00 |
1.22 |
0.94 |
0.95 |
|
||
DMSO control |
Mean |
17.0 |
18.7 |
-- |
101.7 |
-- |
17.3 |
7.7 |
9.0 |
-- |
51.0 |
|
|
MF |
0.96 |
0.92 |
-- |
0.95 |
-- |
1.16 |
0.96 |
1.00 |
-- |
1.00 |
|
||
Distilled water control |
Mean |
17.7 |
20.3 |
83.7 |
106.7 |
17.0 |
15.0 |
8.0 |
9.0 |
47.3 |
51.0 |
|
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
||
500 R/P |
Mean |
17.3 |
18.3 |
40.7 |
96.7 |
15.0 |
11.3 |
6.0 |
6.7 |
49.0 |
49.7 |
|
|
MF |
0.98 |
0.90 |
0.49 |
0.91 |
0.88 |
0.76 |
0.75 |
0.74 |
1.04 |
0.97 |
|
||
158.1 SR/P |
Mean |
17.7 |
19.0 |
61.0 |
109.0 |
16.0 |
16.7 |
3.3 |
5.7 |
50.0 |
51.3 |
|
|
MF |
1.00 |
0.93 |
0.73 |
1.02 |
0.94 |
1.11 |
0.42 |
0.63 |
1.06 |
1.01 |
|
||
50 |
Mean |
16.0 |
17.3 |
84.3 |
103.0 |
14.0 |
15.0 |
7.3 |
7.3 |
48.7 |
51.7 |
|
|
MF |
0.91 |
0.85 |
1.01 |
0.97 |
0.82 |
1.00 |
0.92 |
0.81 |
1.03 |
1.01 |
|
||
15.81 |
Mean |
17.0 |
17.7 |
94.0 |
100.0 |
15.0 |
15.0 |
7.7 |
8.7 |
48.0 |
52.7 |
|
|
MF |
0.96 |
0.87 |
1.12 |
0.94 |
0.88 |
1.00 |
0.96 |
0.96 |
1.01 |
1.03 |
|
||
5 |
Mean |
17.7 |
18.3 |
85.0 |
92.0 |
14.7 |
16.0 |
9.3 |
9.0 |
42.7 |
46.3 |
|
|
MF |
1.00 |
0.90 |
1.02 |
0.86 |
0.86 |
1.07 |
1.17 |
1.00 |
0.90 |
0.91 |
|
||
1.581 |
Mean |
17.0 |
18.7 |
78.0 |
91.0 |
15.3 |
14.3 |
8.7 |
8.7 |
48.0 |
51.0 |
|
|
MF |
0.96 |
0.92 |
0.93 |
0.85 |
0.90 |
0.96 |
1.08 |
0.96 |
1.01 |
1.00 |
|
||
0.5 |
Mean |
16.3 |
18.3 |
80.7 |
111.3 |
14.3 |
14.0 |
8.3 |
8.3 |
48.0 |
49.0 |
|
|
MF |
0.92 |
0.90 |
0.96 |
1.04 |
0.84 |
0.93 |
1.04 |
0.93 |
1.01 |
0.96 |
|
||
0.1581 |
Mean |
17.0 |
18.3 |
85.7 |
94.0 |
16.0 |
15.0 |
7.7 |
9.7 |
45.3 |
51.0 |
|
|
MF |
0.96 |
0.90 |
1.02 |
0.88 |
0.94 |
1.00 |
0.96 |
1.07 |
0.96 |
1.00 |
|
||
NPD (4mg) |
Mean |
417.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
|
MF |
24.55 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
||
2AA (2mg) |
Mean |
-- |
2421.3 |
-- |
2466.7 |
-- |
208.0 |
-- |
205.0 |
-- |
-- |
|
|
MF |
-- |
129.71 |
-- |
24.26 |
-- |
12.00 |
-- |
22.78 |
-- |
-- |
|
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
206.7 |
|
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
4.05 |
|
||
SAZ (2mg) |
Mean |
-- |
-- |
1057.3 |
-- |
1176.0 |
-- |
-- |
-- |
-- |
-- |
|
|
MF |
-- |
-- |
12.64 |
-- |
69.18 |
-- |
-- |
-- |
-- |
-- |
|
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
441.3 |
-- |
-- |
-- |
|
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
57.57 |
-- |
-- |
-- |
|
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1024.0 |
-- |
|
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
21.63 |
-- |
|
||
*Assay3
P: Precipitate
R: Reduced background lawn development
SR: Slightly reduced background lawn development
MF: Mutation factor
SD: Standard deviation
+S9: with S9 mix
-S9: withoutS9 mix
Applicant's summary and conclusion
- Conclusions:
- 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 Saccharomyces cerevisiae cell wall, extractedhad 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. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test, an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-Incubation Method) and an Assay 3 (Pre-Incubation Method). The performed Assay 2 in case of Salmonella typhimuriumTA1535 strain with and without metabolic activation could not be properly evaluated due to the observed contamination on the plates. Therefore, the performed Assay 2 in case of this strain was declared invalid and was repeated in an additional experiment (Assay 3) with and without metabolic activation with the same experimental conditions as in Assay 2. Based on the results of the preliminary experiment, the examined test concentrations in the assays were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581μg/plate.
In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect. Precipitate was detected on the plates in the main tests in all examined bacterial strains with and without metabolic activation at higher concentrations. Inhibitory, cytotoxic effect of the test item on the lawn growth of the bacteria was observed in the main tests in all examined bacterial strains with and without metabolic activation at higher concentrations.
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 Saccharomyces cerevisiae cell wall, extracted had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
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