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EC number: 226-073-0 | CAS number: 5261-99-4
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 3 December 2012 to 8 March 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: OECD guideline and EU method. GLP study.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- [(±)-(4-amino-2-hydroxy-4-oxobutyl)trimethylammonium] chloride
- EC Number:
- 226-073-0
- EC Name:
- [(±)-(4-amino-2-hydroxy-4-oxobutyl)trimethylammonium] chloride
- Cas Number:
- 5261-99-4
- Molecular formula:
- C7H17N2O2.Cl
- IUPAC Name:
- 4-amino-2-hydroxy-N,N,N-trimethyl-4-oxobutan-1-aminium chloride
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- - Physical state: white powder
- Analytical purity: 99,4%
- Lot/batch No.: WI 2274
- Storage condition of test material: controlled room temperature (15-25 ºC, below 70 RH%)
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Initial and confirmatory tests: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene-diamine (NPD)
- Remarks:
- Without metabolic activation: TA98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Without metabolic activation: TA100, TA1535
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without metabolic activation: TA1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Without metabolic activation: E. coli WP2 uvr A
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- With metabolic activation: all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Initial test: standard plate incorporation procedure. 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). 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 (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.
Confirmatory test: pre-incubation procedure. Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture 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 or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.
NUMBER OF REPLICATIONS: Three
DETERMINATION OF CYTOTOXICITY
- Method: The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C. - Evaluation criteria:
- Criteria for a Positive Response:
A test item was considered mutagenic if:
-a dose–related increase in the number of revertants occurred and/or;
-a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
-in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA strains the number of reversion was more than twice higher than the spontaneous reversion rate of the negative (solvent) control plates,
-in Salmonella typhimurium TA1535 and TA1537 strains the number of reversions was more than three times higher than the spontaneous reversion rate of the negative (solvent) control plates.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
The observed revertant colony numbers in Salmonella typhimurium TA100 strain of the preliminary experiment were higher than the upper limit of the historical control range on the test item treated and control plates; however this fact was considered not to adversely affect the dose selection for the main tests.
Slightly higher or slightly lower numbers of revertant colonies compared to the solvent control were detected in the two tester strains at some concentrations. However, they had no biological significance; thus, they were considered as reflecting the variability of the test system.
No insolubility or signs of cytotoxicity was observed in the preliminary experiment.
COMPARISON WITH HISTORICAL CONTROL DATA:
Sporadically, higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No signs of cytotoxicity were observed in the main tests. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 1: Summary Table of the Initial Mutation Test
Concentrations |
Mean |
Salmonella typhimurium tester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
23.0 |
35.0 |
91.0 |
119.7 |
7.3 |
10.0 |
12.7 |
11.0 |
35.3 |
37.7 |
MF |
0.96 |
1.01 |
0.93 |
1.02 |
1.22 |
1.07 |
1.52 |
0.87 |
0.75 |
0.77 |
|
DMSO control |
Mean |
23.3 |
34.3 |
-- |
109.3 |
-- |
10.7 |
11.7 |
11.0 |
-- |
25.7 |
MF |
0.97 |
0.99 |
-- |
0.93 |
-- |
1.14 |
1.40 |
0.87 |
-- |
0.52 |
|
Distilled water control |
Mean |
24.0 |
34.7 |
97.7 |
117.7 |
6.0 |
9.3 |
8.3 |
12.7 |
47.0 |
49.0 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
27.0 |
29.0 |
92.3 |
122.3 |
12.3 |
8.7 |
11.0 |
14.3 |
46.7 |
50.7 |
MF |
1.13 |
0.84 |
0.95 |
1.04 |
2.06 |
0.93 |
1.32 |
1.13 |
0.99 |
1.03 |
|
1581 |
Mean |
27.0 |
38.3 |
95.7 |
119.3 |
6.7 |
8.3 |
9.3 |
9.7 |
47.3 |
47.3 |
MF |
1.13 |
1.11 |
0.98 |
1.01 |
1.11 |
0.89 |
1.12 |
0.76 |
1.01 |
0.97 |
|
500 |
Mean |
25.3 |
32.3 |
97.3 |
109.7 |
5.7 |
9.7 |
11.3 |
10.3 |
48.7 |
38.3 |
MF |
1.06 |
0.93 |
1.00 |
0.93 |
0.94 |
1.04 |
1.36 |
0.82 |
1.04 |
0.78 |
|
158.1 |
Mean |
25.7 |
30.0 |
98.7 |
111.7 |
9.0 |
9.0 |
12.3 |
11.7 |
35.7 |
48.0 |
MF |
1.07 |
0.87 |
1.01 |
0.95 |
1.50 |
0.96 |
1.48 |
0.92 |
0.76 |
0.98 |
|
50 |
Mean |
32.0 |
32.7 |
98.0 |
104.7 |
4.3 |
11.0 |
15.0 |
10.7 |
40.7 |
51.7 |
MF |
1.33 |
0.94 |
1.00 |
0.89 |
0.72 |
1.18 |
1.80 |
0.84 |
0.87 |
1.05 |
|
15.81 |
Mean |
24.3 |
37.3 |
100.7 |
124.0 |
7.7 |
8.7 |
12.3 |
8.3 |
40.3 |
44.3 |
MF |
1.01 |
1.08 |
1.03 |
1.05 |
1.28 |
0.93 |
1.48 |
0.66 |
0.86 |
0.90 |
|
5 |
Mean |
21.7 |
26.0 |
102.3 |
117.3 |
6.3 |
6.7 |
8.3 |
12.3 |
36.3 |
35.7 |
MF |
0.90 |
0.75 |
1.05 |
1.00 |
1.06 |
0.71 |
1.00 |
0.97 |
0.77 |
0.73 |
|
NPD (4µg) |
Mean |
320.0 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
13.71 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2263.0 |
-- |
2828.0 |
-- |
250.7 |
-- |
253.7 |
-- |
-- |
MF |
-- |
65.91 |
-- |
25.87 |
-- |
23.50 |
-- |
23.06 |
-- |
-- |
|
2AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
293.0 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
11.42 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1700.7 |
-- |
656.3 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
17.41 |
-- |
109.39 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
380.0 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
32.57 |
-- |
-- |
-- |
|
MMS (2µL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
988.3 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
21.03 |
-- |
|
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item had no mutagenic activity on the growth of the applied bacterium tester 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 TA98, TA100, TA1535 and TA1537 and the tryptophan-requiring auxotroph strain of Escherichia coli WP2 uvrA in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. Based on the results of the Solubility Test, the test item was dissolved in Distilled water. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normalbiological variability of the test system. The mean values of revertant colonies of the 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 tests were considered to be valid. In conclusion, the test item had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.
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