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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.02.2000-29.03.2000
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)
EC Number:
916-222-4
Molecular formula:
C51H99N2O6.CH3O4S
IUPAC Name:
Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)
additive 1
Chemical structure
Reference substance name:
Propane-1,2-diol
EC Number:
200-338-0
EC Name:
Propane-1,2-diol
Cas Number:
57-55-6
Molecular formula:
C3H8O2
IUPAC Name:
propylene glycol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 134428

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 C on the day of each experiment. An allowance for test material purity (94%) was made prior to formulation for the first and second experiments. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use , the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2mm pellets with a nominal pore diameter of 4x10^-4 microns.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Salmonella strains with S9: 5,15,50,150, 500 and 1500 ug/plate
Salmonella strains without S9: 1.5, 5, 15, 50, 150 and 500 ug/plate
Escherichia coli strain WP2uvrA with and without S9: 15,50, 150, 500, 1500 and 5000 ug/plate

Justification for top dose: toxicity. Minimum of four non-toxic doses were plated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 1 to 9.9 x 10^9 per mL

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1 to 9.9 x 10^9 per mL
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett´s method of linear regression(5)) significant increase in the recertant count in at least one strain of bacteria.
Statistics:
Dunnett's method

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Any other information on results incl. tables

The number of revertant colonies for the toxicity assay
With (+) or without (-) S9-mix Strain Dose (µg/plate)
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 127 141 131 132 143 115 108 595 0V C 0T
+ TA100 121 99 109 118 126 126 143 136 118 0T 0T
- WP2uvrA 23 22 19 19 17 17 22 26 25 125 0T
+ WP2uvrA 18 26 25 23 25 23 26 37 24 19 0V
C = contaminated    
S = sparse bacterial background lawn
T = toxic, no bacterial background lawn
V = very weak bacterial background lawn

EXP 1
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
143   16   31   40   9  
167 (150) 22 (20) 28 (27) 23 (30) 9 (12)
139   23   22   26   17  

EXP 2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
148   12   28   13   7  
125 (136) 18 (14) 44 (36) 15 (16) 13 (10)
136   13   37   21   11  

TABLE 2
With or without S9-mix Test substance concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
- 0 170 (154) 20 (25) 34 (24) 28 (28) 13 (12)
136 17.0# 31 5.5 20 8.7 25 3.0 8 3.2
155   25   18   31   14  
- 1.5 117 (130) 19 (22) N/T 25 (32) 11 (10)
145 14.1 26 3.8 38 6.5 11 2.3
128   20   32   7  
- 5 127 (136) 21 (17) N/T 22 (29) 13 (10)
136 8.5 14 3.6 27 8.7 11 3.1
144   16   39   7  
- 15 146 (143) 15 (21) 22 (22) 28 (31) 15 (14)
141 2.5 25 5.3 22 0.6 36 4.6 13 1.0
143   23   21   28   14  
- 50 115 (134) 24 (27) 21 (23) 36 (26) 11 (14)
139 17.5 25 4.9 27 3.5 17 9.5 21 5.8
149   33   21   26   11  
- 150 48 (57) 22 (18) 34 (26) C (24) 17 (16)
55 9.6 14 4.0 25 7.5 27 4.9 11 4.6
67   19   19   20   20  
- 500 0V (0) 0V (0) 22 (21) 18S (14) 0V (0)
0V 0.0 0V 0.0 22 2.3 9S 4.6 0V 0.0
0V   0V   18   15S   0V  
- 1500 N/T N/T 0V (0) N/T N/T
0V 0.0
0V  
- 5000 N/T N/T 0T (0) N/T N/T
0T 0.0
0T  
Positive controls

S9-Mix

-		 Name ENNG ENNG ENNG 4NQO 9AA
Concentration (µg/plate) 3 5 2 0.2 80
No. Colonies per plate 412 (396) 153 (154) 382 (374) 131 (123) 1085 (980)
370 22.5 156 2.1 344 26.9 102 18.0 1082 179.3
405   152   396   135   773  
ENNG N-ethyl-N´-nitro-N-nitrosoguanidine       N/T Not tested at this dose level  
4NQO 4-Nitroquinoline-1-oxide         S Sparse bacterial background lawn
# Standard deviation         T Toxic, no bacterial background lawn
C Contaminated           V Very weak bacterial background lawn

TABLE 3
With or without S9-mix Test substnace concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
+ 0 154 (151) 10 (15) 26 (25) 39 (34) 19 (15)
137 13.2# 19 4.7 22 3.1 36 6.8 15 4.0
163   17   28   26   11  
+ 5 162 (153) 11 (13) N/T 37 (40) 14 (15)
156 10.3 14 2.1 49 7.9 17 2.1
142   15   34   13  
+ 15 143 (138) 18 (15) 25 (23) 31 (33) 9 (11)
132 5.7 13 2.6 21 2.1 39 4.9 12 1.5
140   14   24   30   11  
+ 50 161 (151) 18 (17) 24 (22) 39 (36) 14 (13)
138 11.8 17 1.5 21 2.1 42 8.5 16 4.2
154   15   20   26   8  
+ 150 144 (155) 10 (12) 20 (19) 41 (43) 13 (14)
163 9.8 8 5.3 21 2.6 44 1.7 12 3.2
158   18   16   44   18  
+ 500 22 (17) 11 (9) 23 (25) 41 (40) 10 (10)
16 4.6 4 4.7 26 1.5 40 1.5 14 4.0
13   13   25   38   6  
+ 1500 0V (0) 0V (0) 23V (21) 0V (0) 0V (0)
0V 0 0V 0 17V 3.8 0V 0 0V 0
0V   0V   24V   0V   0V  
+ 5000 N/T N/T 0T (0) N/T N/T
0T 0.0
0T  
Positive controls

S9-Mix

+		 Name 2AA 2AA 2AA BP 2AA
Concentration (µg/plate) 1 2 10 5 2
No. Colonies per plate 2212 (1876) 301 (307) 1001 (929) 407 (376) 513 (584)
1695 291.0 296 14.9 877 31.0 376 31.0 619 61.2
1722   324   908   345   619  
BP Benzo(a)pyrene         N/T Not tested at this dose level  
2AA 2-Aminoanthracene         T Toxic, no bacterial background lawn  
# Standard deviation         V Very weak bacterial background lawn  

TABLE 4
With or without S9-mix Test substnace concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
- 0 149 (138) 15 (17) 35 (43) 23 (20) 5 (8)
125 12.2# 20 2.9 56 11.2 15 4.2 9 3.1
141   15   39   21   11  
- 1.5 137 (144) 22 (18) N/T N/T 5 (8)
159 13.0 20 5.3 11 3.1
136   12   9  
- 5 148 (138) 19 (17) N/T 22 (21) 16 (10)
133 8.4 15 2.1 20 1.2 7 5.2
134   16   20   7  
- 15 124 (141) 21 (17) 34 (33) 18 (19) 16 (11)
151 14.8 15 3.2 32 1.2 26 6.1 10 4.6
148   16   32   14   7  
- 50 158 (151) 19 (18) 34 (35) 21 (20) 10 (8)
155 10.2 20 2.6 36 1.2 21 1.7 7 1.5
139   15   36   18   7  
- 150 81 (71) 11 (11) 36 (38) 24 (24) 11 (9)
76 12.7 6 5.0 35 3.8 23 0.6 8 1.5
57   16   42   24   9  
- 500 0V (0) 0V (0) 32 (39) 9S (9) 0V (0)
0V 0.0 0V 0.0 45 6.6 11S 1.5 0V 0.0
0V   0V   40   8S   0V  
- 1500 N/T N/T 0V (0) 0V (0) N/T
0V 0.0 0V 0.0
0V   0V  
- 5000 N/T N/T 0T (0) N/T N/T
0T 0.0
0T  
Positive controls

S9-Mix

-		 Name ENNG ENNG ENNG 4NQO 9AA
Concentration (µg/plate) 3 5 2 0.2 80
No. Colonies per plate 393 (413) 298 (299) 737 (740) 104 (105) 1323 (1399)
396 32.7 284 16.0 755 14.2 110 4.2 1392 79.7
451   316   727   102   1482  
ENNG N-ethyl-N´-nitro-N-nitrosoguanidine N/T Not tested at this dose level
4NQO 4-Nitroquinoline-1-oxide S Sparse bacterial background lawn
# Standard deviation T Toxic, no bacterial background lawn
C Contaminated V Very weak bacterial background lawn

TABLE 5
With or without S9-mix Test substnace concentration (µg/plate) Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
+ 0 98 (124) 13 (12) 37 (44) 36 (34) 16 (13)
148 25.1# 12 0.6 56 10.2 36 4.0 16 5.2
126   12   40   29   7  
+ 5 170 (148) 10 (11) N/T 32 (32) 15 (13)
137 19.1 12 1.2 32 0.6 15 3.5
137   10   33   9  
+ 15 130 (134) 11 (10) 36 (39) 36 (34) 13 (12)
143 7.5 8 1.7 45 4.9 35 2.1 11 1.0
130   11   37   32   12  
+ 50 148 (146) 20 (16) 36 (37) 24 (26) 13 (13)
162 16.6 16 4.5 36 2.3 24 3.5 17 3.5
129   11   40   30   10  
+ 150 143 (146) 10 (11) 48 (36) 32 (29) 14 (12)
127 20.7 13 1.5 23 12.6 32 5.2 18 6.7
168   11   38   23   5  
+ 500 31 (29) 13 (9) 41 (41) 31 (28) 6 (8)
23 5.7 8 3.2 46 4.5 30 3.8 9 1.5
34   7   37   24   8  
+ 1500 0V 0 0V 0 35 (38) 0V 0 0V 0
0V 0.0 0V 0.0 32 8.5 0V 0.0 0V 0.0
0V   0V   48   0V   0V  
+ 5000 N/T N/T 0T (0) N/T N/T
0T 0.0
0T  
Positive controls

S9-Mix

+		 Name 2AA 2AA 2AA BP 2AA
Concentration (µg/plate) 1 2 10 5 2
No. Colonies per plate 1722 (1659) 334 (325) 622 (586) 297 (289) 518 (390)
1744 128.6 350 31.8 572 31.8 261 25.4 345 112.1
1511   291   563   310   308  
BP Benzo(a)pyrene N/T Not tested at this dose level
2AA 2-Aminoanthracene T Toxic, no bacterial background lawn
# Standard deviation V Very weak bacterial background lawn

Historical Profile - Vehicle Control Values 1998
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9
Mean  124 125 31 36 26 18 26 28 11 14 23 25 262 285
SD 16 16 6 6 5 4 6 5 3 3 4 4 31 32
Min 73 81 15 22 12 10 15 18 5 6 13 13 191 231
Max 173 170 45 54 38 37 26 26 20 23 34 41 304 363
Values 342 339 337 340 341 331 16 16 323 325 287 285 20 22

Historical Profile - Positive Control Values 1998
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9
Mean 556 1209 143 472 375 260 403 556 1052 298 851 837 863 747
SD 114 318 24 119 173 64 93 197 259 106 202 223 127 116
Min 349 681 101 198 112 105 305 344 173 123 415 260 646 591
Max 1147 2740 225 733 1143 601 604 1073 1912 652 1486 1380 1096 1096
Values 141 141 140 143 142 141 12 12 142 141 138 138 24 25

Historical Profile - Vehicle Control Values 1999
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9
Mean 102 107 24 31 22 16 13 19 11 17 23 27 263 298
SD 19 20 6 7 6 4 5 5 3 4 5 6 42 32
Min 57 63 10 12 10 7 5 8 3 5 10 11 188 222
Max 179 186 46 56 40 35 32 31 25 26 52 46 352 363
Values 751 592 646 510 645 488 57 31 634 481 641 495 89 37

Historical Profile - Positive Control Values 1999
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9 -S9 + S9
Mean 549 1019 146 312 368 245 327 446 1017 345 787 598 882 737
SD 176 378 32 129 237 60 157 184 307 152 251 232 186 117
Min 238 406 95 113 104 123 113 215 350 112 200 208 583 509
Max 1170 2060 290 717 1229 467 653 837 2031 870 1760 1294 1673 1261
Values 161 160 162 160 158 157 27 27 159 158 153 152 52 51

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirement of the OECD Guidelines for Testing of Chemicals No. 471 "Reverse Mutation Study", Method B14 of Commision Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The dose range for the first experiment was determined in a preliminary toxicity assay and was 1.5 to 500 and 5 to 1500 µg/plate for the Salmonella strains without and with S9 respectively. The dose range for Escherichia coli strain WP2uvrA was 15 to 5000 µg/plate. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels (in excess of the five specified in the standard test method) were employed to allow for the toxicity of the test material and to ensure there were a minimum of four non toxic doses plated out.

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 and with metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused both a visible reduction in the growth of the bacterial lawn and/or a significant decrease in the frequency of revertant colonies to all of the tester strains both with and without S9 -mix. The first incidence of a weakened lawn was observed to the Salmonella strains at 500 µg/plate and Escherichia coli strain WP2uvrA at 1500 µg/plate. the test material was, therefore, tested up to the toxic limit. No test material 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 material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.