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EC number: 225-266-7 | CAS number: 4747-21-1
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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 008
- Report date:
- 2008
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (1997)
- Deviations:
- yes
- Remarks:
- only 2-AA was used as only positive control substance in presence of S9-mix.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- (2000)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- (1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N-methylisopropylamine
- EC Number:
- 225-266-7
- EC Name:
- N-methylisopropylamine
- Cas Number:
- 4747-21-1
- Molecular formula:
- C4H11N
- IUPAC Name:
- methyl(propan-2-yl)amine
- Details on test material:
- - Name of test material (as cited in study report): N-Methylisopropylamine
- Physical state: liquid, yellowish
- Analytical purity: > 99.9 area% (see analytical report, study code 07L00393)
- Lot/batch No.: 8078/06/056
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility
- Storage condition of test material: room temperature (protected from light)
Constituent 1
Method
- Target gene:
- his operon (for S. typhimurium strains) and trp operon (for E. coli strains).
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:
- cofactors supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- 1st Experiment (all strains, standard plate test with and without S-9 mix, 3 plates/dose): 0; 20; 100; 500; 2500 and 5000 µg/plate
2nd Experiment (all strains, preincubation test with and without S-9 mix, 3 plates/dose): 0; 200; 400; 800; 1600 and 3200 µg/plate
3rd Experiment (all strains, preincubation test without S-9 mix, 3 plates/dose): 0; 10; 50; 250; 1250; 2500 µg/plate
3rd Experiment (all strains, preincubation test with S-9 mix, 3 plates/dose): 0; 20; 100; 500; 2500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Remarks:
- (sterility control)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (water)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: with S9-mix
- Remarks:
- 2-aminoanthracene (2-AA): 2.5 µg/plate dissolved in DMSO for strains TA 1535, TA 100, TA 1537 and TA 98; 60 µg/plate dissolved in DMSO for Escherichia coli WP2 uvrA
- Positive controls:
- yes
- Positive control substance:
- other: without S9-mix
- Remarks:
- N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): 5 µg/plate (DMSO), TA1535, TA100; 4-nitro-o-phenylendiamine (NOPD): 10 µg/plate (DMSO), TA98; 9-aminoacridine (AAC): 100 µg/plate (DMSO), TA1537; 4-nitroquinoline-N-oxide (4-NQO): 5 µg/plate (DMSO), WP2uvrA
- Details on test system and experimental conditions:
- TEST DESIGN
Standard plate test (SPT) and preincubation test (PIT), both with and without metabolic activation, were performed.
Standard plate test (SPT, Experiment 1):
The experimental procedure was based on Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron & Ames (Mut. Res. 113: 173-215, 1983).
Test tubes containing 2 mL portions of soft agar [100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin for S. typhimurium or 0.5 mM tryptophan for E.coli)] were kept in a water bath at about 42 - 45°C. 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture and 0.5 mL S9 mix (in case of metabolic activation) or 0.5 mL phosphate buffer (in case of no metabolic activation) were added.
After mixing, the samples were poured onto agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.
Preincubation Test (PIT, Experiment 2 and 3):
The experimental procedure was based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980). 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (in case of metabolic activation) or phosphate buffer (in case of no metabolic activation) were incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.
PARAMETERS EXAMINED
Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
Titer:
The titer was determined only in the experimental parts with S9 mix both for the
negative controls (vehicle only) and for the two highest doses in all experiments.
Cytotoxicity:
Toxicity was detected by (1) decrease in the number of revertants, (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth) and (3) reduction in the titer. Cytotoxicity was recorded for all test groups both with and without S9 mix in all experiments.
Solubility:
Precipitation of the test item was recorded and indicated. As long as precipitation does not interfere with colony scoring, 5000 µg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. - Evaluation criteria:
- Acceptance criteria:
The experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain;
- The sterility controls reveales no indication of bacterial contamination;
- The positive control substances both with and without S9 mix induce a distinct increase in number of revertant colonies within the range of the historical positive control data or above;
- The titer of viable bacteria is egal to/greater than 10E+8/mL.
Assessment criteria:
The test item is positive if a dose-related and reproducible increase in the number of revertant colonies, i .e . about doubling of the spontaneous mutation rate in at least one tester strain either without or with S9-mix, is observed.
The test item is generally nonmutagenic if the number of revertants for all tester strains is within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
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
- Remarks:
- no increases in his+ revertant colonies were observed
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- depending on strain and test conditions, from 2500 µg/plate onward in SPT and from 1250 µg/plate onward in PIT
- 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
- Remarks:
- no increases in trp+ revertant colonies were observed
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- depending on strain and test conditions, from 2500 µg/plate onward in SPT and from 1250 µg/plate onward in PIT
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MUTAGENICITY
When tested in the S. typhimurium strains TA1535, TA1537, TA100 and TA98 and in E. coli WP2uvrA under standard plate test (SPT) and preincubation test (PIT) conditions, with and without S9 mix, the test item did not induce an increase in his+ or trp+ revertant colonies at concentrations up to 5000 µg/plate.
CYTOTOXICITY
A cytototoxic effect was observed in the SPT depending on the strain and test conditions from 2 500 μg/plate onward; iIn the PIT, cytotoxicity was observed from 1250 µg/plate onward.
PRECIPITATION
No test item precipitation was observed within the concentration range tested, with and without S9-mix.
CONTROLS
The number of revertant colonies in the negative controls was within the range of the historical negative control data reported in the study for each tester strain, both with and without S9-mix. The positive control substances induced the expected increased in revertant colonies, both with and without S9-mix; the results were within the historical positive control range. Thus, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. - Remarks on result:
- other: other: standard plate test (SPT, Experiment 1) and preincubation tests (PIT; Experiments 2 and 3)
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
N-Methylisopropylamine: Bacterial Reverse Mutation Assay, mean revertants colonies/plate |
||||||||||||||
EXPERIMENT 1 (Standard Plate Test, SPT) |
||||||||||||||
Strain |
S. typhimurium strains |
|||||||||||||
TA1535 |
TA100 |
TA1537 |
TA98 |
|||||||||||
Test item (µg/plate) |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||
NC*** |
13(1)* |
14(1) |
95(1) |
98(1) |
9(1) |
9(1) |
31(1) |
44(1) |
||||||
20 |
12(0.9) |
16(1.1) |
100(1) |
101(1) |
7(0.7) |
9(1.1) |
30(1) |
50(1.1) |
||||||
100 |
12(0.9) |
15(1.1) |
91(1) |
97(1) |
8(0.9) |
10(1.2) |
35(1.1) |
52(1.2) |
||||||
500 |
14(1.1) |
14(1.0) |
95(1) |
92(0.9) |
7(0.8) |
6(0.7) |
37(1.2) |
46(1) |
||||||
2500 |
9(0.7) |
11(0.8) |
76(0.8) |
71(0.7) |
6(0.7) |
6(0.7) |
19(0.6) |
27(0.6) |
||||||
5000 |
0(RB)** |
0(RB) |
0(RB) |
RB/20(0.2) |
0(RB) |
0(RB) |
0(RB) |
0(RB) |
||||||
PC*** |
578(44.5) |
135(9.9) |
629(6.6) |
635(6.5) |
360(40) |
137(15.8) |
486(15.7) |
549(12.4) |
||||||
Strain |
E.coli WP2uvrA |
*, the mutation factor is given in brackets **, RB = reduced background growth indicating cytotoxicity ***, NC = negative (vehicle) control; PC = respective positive control |
||||||||||||
Test item (µg/plate) |
-S9 |
+S9 |
||||||||||||
NC |
31(1) |
33(1) |
||||||||||||
20 |
29(0.9) |
32(1) |
||||||||||||
100 |
30(1) |
39(1.2) |
||||||||||||
500 |
29(0.9) |
40(1.2) |
||||||||||||
2500 |
21(0.7) |
22(0.7) |
||||||||||||
5000 |
0(RB) |
RB/15(0.4) |
||||||||||||
PC |
567(18.3) |
258(7.7) |
||||||||||||
EXPERIMENT 2 (Preincubation Test, PIT) |
||||||||||||||
Strain |
S. typhimurium strains |
|||||||||||||
TA1535 |
TA100 |
TA1537 |
TA98 |
|||||||||||
Test item (µg/plate) |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||
NC*** |
18(1)* |
19(1) |
99(1) |
111(1) |
9(1) |
9(1) |
28(1) |
28(1) |
||||||
200 |
21(1.1) |
21(1.1) |
94(0.9) |
125(1.1) |
7(0.7) |
8(0.9) |
22(0.8) |
33(1.2) |
||||||
400 |
16(0.9) |
24(1.3)**** |
110(1.1) |
122(1.1) |
8(0.9) |
8(0.9) |
20(0.7) |
34(1.2) |
||||||
800 |
20(1.1) |
23(1.3) |
103(1) |
133(1.2) |
8(0.9) |
9(1) |
23(0.8) |
36(1.3) |
||||||
1600 |
0(RB)** |
19(1) |
0(RB) |
120(1.1) |
0(RB) |
6(0.7) |
0(RB) |
27(0.6) |
||||||
3200 |
0(RB)** |
16 (0.9) |
0(RB) |
118(1.1) |
0(RB) |
6(0.7) |
0(RB) |
26(0.9) |
||||||
PC |
559(31) |
119(6.4) |
616(6.2) |
552(5) |
348(38.7) |
133(15.3) |
443(15.6) |
597(21.3) |
||||||
PIT (Exp.2) |
E.coli WP2uvrA |
*, the mutation factor is given in brackets **, RB = reduced background growth indicating cytotoxicity ***, NC = negative (vehicle) control; PC = respective positive control ****, because of contamination. only 2/3 plates evaluated |
||||||||||||
Test item (µg/plate) |
-S9 |
+S9 |
||||||||||||
NC |
34(1) |
44(1) |
||||||||||||
200 |
40(1.2) |
45(1) |
||||||||||||
400 |
34(1) |
42(1) |
||||||||||||
800 |
36(1.1) |
45(1) |
||||||||||||
1600 |
0(RB) |
30(0.7) |
||||||||||||
3200 |
0(RB) |
33(0.7) |
||||||||||||
PC |
729(21.2) |
199(4.6) |
||||||||||||
EXPERIMENT 3 (Preincubation Test, PIT) |
||||||||||||||
S9 -mix |
Without
|
|||||||||||||
Test item (µg/plate) |
TA1535 |
TA100 |
TA1537 |
TA98 |
E.coli WP2uvrA |
|||||||||
NC |
17(1) |
102(1) |
9(1) |
27(1) |
40(1) |
|||||||||
10 |
15(0.9) |
92(0.9) |
7(0.7) |
28(1.1) |
36(0.9) |
|||||||||
50 |
18(1.1) |
104(1) |
8(0.9) |
23(0.9) |
38(1.0) |
|||||||||
250 |
14(0.8) |
101(1) |
6(0.7) |
23(0.9) |
32(0.8) |
|||||||||
1250 |
6(0.4)/RB) |
64(0.6) |
4(0.4)/RB |
19(0.7) |
24(0.6) |
|||||||||
2500 |
0(RB) |
0(RB) |
0(RB) |
0(RB) |
0(RB) |
|||||||||
PC |
753(44.3) |
670(6.7) |
481(51.5) |
464(17.4) |
634(15.7) |
|||||||||
S9-mix
|
With |
|||||||||||||
Test item (µg/plate) |
TA1535 |
TA100 |
TA1537 |
TA98 |
E.coli WP2uvrA |
|||||||||
NC |
17(1) |
101(1) |
8(1) |
33(1) |
47(1) |
|||||||||
20 |
17(1) |
105(1) |
11(1.3) |
37(1.1) |
43(0.9) |
|||||||||
100 |
15(0.9) |
100(1) |
8(1) |
34(1) |
44(0.9) |
|||||||||
500 |
18(1.1) |
97(1) |
8(1) |
31(0.9) |
41(0.9) |
|||||||||
2500 |
21(1.2) |
91(0.9) |
8(1) |
32(1) |
42(0.9) |
|||||||||
5000 |
12 (0.7) |
88(0.9) |
5(0.6) |
21(0.6) |
37(0.8) |
|||||||||
PC |
142(8.5) |
824(8.1) |
154 (18.5) |
891(26.7) |
221(4.7) |
|||||||||
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
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