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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:
24 Jul 2017 - 21 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP-Landesleitstelle Bayern, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts
EC Number:
500-232-7
EC Name:
Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts
Cas Number:
68815-56-5
Molecular formula:
not applicable to UVCB
IUPAC Name:
Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

Method

Target gene:
His operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavoneinduced male rats
Test concentrations with justification for top dose:
Experiment I (the incorporation test): 3.16, 10.0, 31.6 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II (the pre-incubation test): 3.16, 10.0, 31.6 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest., Eurofins Munich, Lot No. 170710, 170804

Controls
Untreated negative controls:
yes
Remarks:
A. dest.
Negative solvent / vehicle controls:
yes
Remarks:
A. dest.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 10 μg/plate for TA 98, 40 μg/plate for TA 1537, without metabolic activation; 2-aminoanthracene, 2.5 μg/plate for TA 98, TA 100, TA 1535 and TA 1537, 10 μg/plate for TA 102, with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 – 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: the diminution of the background lawn or a significant reduction in the number of revertants in solvent control.
Evaluation criteria:
Negative control data within historical range. Positive controls show a distinct enhancement of revertant rates over the control plate. At least five different concentrations of each tester strain are analyzable.
Positive response: a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as:
- if in tester strains TA 98 and TA 100 the number of reversions is at least and twice as high
- if in tester strains TA 102 the number of reversions is at least 1.5-fold higher
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Negative response: neither a dose related increase in the number of revertants nor a reproducible biologically relevant increase.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 102 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A pre-experiment for toxicity was performed at 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with tester strains TA 98 and TA 100. The test substance were observed toxic effects at the concentration of 1000 μL/plate or higher.
HISTORICAL CONTROL DATA
- Positive historical control data: Positive controls were met the criteria of validity
- Negative (solvent/vehicle) historical control data: Negative controls were within historical norms

Any other information on results incl. tables

Table 1: Results Experiment I

TesterStrain:TA98                                                                                       Experiment:1

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 24 27 3 41 37 5.9 1 1
27 30
30 39
Test Item 3.16 µg 21 27 9.3 26 32 5.3 1 0.9
23 34
38 36
Test Item 10.0 µg 23 27 3.5 38 36 4.7 1 1
30 40
27 31
Test Item 31.6 µg 16 20 3.5 38 34 3.6 0.7 0.9
23 31
20 33
Test Item 100 µg 24 31 6.4 39 32 7.5 1.2 0.9
36 24
34 33
Test Item 316 µg 29 24 4.4 37 34 4.4 0.9 0.9
22 29
21 36
Test Item 1000 µg 16 18 1.5 25 27 2 0.7 0.7
19 27
18 29
Test Item 2500 µg 0 B 0 0 13 B 17 6.4 0 0.5
0 B 13 B
0 B 24 B
Test Item 5000 µg 0 N 0 0 0 N 0 0 0 0
0 N 0 N
0 N 0 N
4-NOPD 10 µg 482 454 25 / / / 16.8 /
434 /
446 /
2-AA 2.5 µg / / / 2419 2465 225 / 67.2
/ 2266
/ 2709

SD: Standard-deviation;  P: Precipitation B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor = mean revertants (test item)/ mean revertants (vehicle control)

TesterStrain:TA100                                                                                      Experiment:1

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 82 104 105 84 106 107 108 109
117 83
112 87
Test Item 3.16 µg 79 83 3.5 84 84 0 0.8 1
83 84
86 84
Test Item 10.0 µg 111 102 11.7 94 101 7 1 1.2
89 108
107 102
Test Item 31.6 µg 102 89 11 107 112 7.6 0.9 1.3
82 121
84 109
Test Item 100 µg 114 94 17.3 80 94 17.6 0.9 1.1
83 114
85 89
Test Item 316 µg 81 74 5.8 82 88 10.4 0.7 1
71 100
71 82
Test Item 1000 µg 34 B 44 9.5 67 B 64 3.5 0.4 0.8
44 B 64 B
53 B 60 B
Test Item 2500 µg 34 B 34 1.5 57 B 59 2 0.3 0.7
33 B 59 B
36 B 61 B
Test Item 5000 µg 0 N 0 0 59 B 68 8.6 0 0.8
0 N 70 B
0 N 76 B
NaN3 10 µg 729 714 42.5 / / / 6.9 /
747 /
666 /
2-AA 2.5 µg / / / 1834 1927 80.8 / 22.8
/ 1982
/ 1964

TesterStrain:TA1535                                                                                    Experiment:1

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 16 17 18 7 10 3 1 1
15 10
19 13
Test Item 3.16 µg 22 17 4.4 8 9 1.2 1 0.9
15 10
14 8
Test Item 10.0 µg 16 18 4.7 8 8 0.6 1.1 0.8
14 8
23 7
Test Item 31.6 µg 15 15 3 10 10 2 0.9 1
18 12
12 8
Test Item 100 µg 15 15 3 14 11 3.5 0.9 1.1
18 7
12 11
Test Item 316 µg 4 10 5.1 14 10 4 0.6 1
14 10
11 6
Test Item 1000 µg 3 3 0.6 7 7 1.5 0.2 0.7
4 6
3 9
Test Item 2500 µg 1 B 1 0.6 2 B 4 2.5 0.1 0.4
2 B 7 B
1 B 4 B
Test Item 5000 µg 1 B 1 0.6 3 B 4 1 0.1 0.4
2 B 4 B
1 B 5 B
NaN3 10 µg 772 815 63.3 / / / 48.9 /
888 /
786 /
2-AA 2.5 µg / / / 169 168 9 / 16.8
/ 177
/ 159

TesterStrain:TA1537                                                                                    Experiment:1

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 3 6 3.1 15 8 6.1 1 1
9 7
7 3
Test Item 3.16 µg 11 11 0 11 9 4.4 1.7 1.1
11 12
11 4
Test Item 10.0 µg 8 8 0.6 9 9 1 1.3 1.1
8 10
9 8
Test Item 31.6 µg 15 10 4.7 15 12 2.5 1.5 1.5
8 10
6 12
Test Item 100 µg 10 9 1.7 10 11 1.2 1.4 1.3
10 10
7 12
Test Item 316 µg 12 8 4 10 10 6 1.3 1.2
8 4
4 16
Test Item 1000 µg 9 B 5 3.2 12 14 2.9 0.8 1.6
4 B 12
3 B 17
Test Item 2500 µg 4 B 5 4 7 B 6 1.2 0.7 0.8
9 B 5 B
1 B 7 B
Test Item 5000 µg 3 B 4 2.3 4 B 6 2.1 0.7 0.8
3 B 7 B
7 B 8 B
 4-NOPD 40 µg 96 81 13.7 / / / 12.8 /
69 /
79 /
2-AA 2.5 µg / / / 253 302 52.9 / 36.2
/ 358
/ 294

TesterStrain:TA102                                                                                      Experiment:1

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 331 304 24.3 326 320 7.2 1 1
284 322
297 312
Test Item 3.16 µg 294 283 15.9 398 387 30 0.9 1.2
265 353
291 410
Test Item 10.0 µg 292 295 8.3 394 391 47.6 1 1.2
288 437
304 342
Test Item 31.6 µg 310 322 20.5 395 392 3.6 1.1 1.2
311 393
346 388
Test Item 100 µg 332 313 16.9 368 371 25.1 1 1.2
301 397
305 347
Test Item 316 µg 314 328 14.5 349 319 32.7 1.1 1
343 323
328 284
Test Item 1000 µg 308 323 18.7 387 343 38.8 1.1 1.1
317 326
344 315
Test Item 2500 µg 260 267 19.5 284 315 27.2 0.9 1
252 324
289 336
Test Item 5000 µg 323 323 0.6 284 277 10.4 1.1 0.9
324 282
323 265
MMS 1 µL 2133 1589 552.2 / / / 5.2 /
1029 /
1605 /
2-AA 2.5 µg / / / 727 811 82.1 / 2.5
/ 891
/ 816

Table 3: Results Experiment II

 TesterStrain:TA98                                                                                 Experiment:2

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 26 21 4.6 23 27 3.2 1 1
20 28
17 29
Test Item 3.16 µg 24 24 4.5 25 23 7.2 1.1 0.9
28 29
19 15
Test Item 10.0 µg 21 20 1.5 17 20 4.2 0.9 0.8
18 25
20 19
Test Item 31.6 µg 35 28 15.7 32 28 4.6 1.3 1.1
39 29
10 23
Test Item 100 µg 18 21 3.6 28 24 4 1 0.9
25 20
20 23
Test Item 316 µg 22 19 2.9 26 26 0 0.9 1
17 26
17 26
Test Item 1000 µg 14 B 13 1.2 18 20 2.5 0.6 0.8
14 B 20
12 B 23
Test Item 2500 µg 10 B 7 3.6 15 B 16 1.7 0.3 0.6
3 B 18 B
8 B 15 B
Test Item 5000 µg 0 N 0 0 8 B 11 5.5 0 0.4
0 N 17 B
0 N 7 B
4-NOPD 10 µg 280 287 6.7 / / / 13.7 /
293 /
289 /
2-AA 2.5 µg / / / 2127 1954 169.2 / 73.3
/ 1945
/ 1789

TesterStrain:TA100                                                                                      Experiment:2

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 117 102 23.7 81 80 4 1 1
115 76
75 84
Test Item 3.16 µg 82 86 5.9 98 90 9.8 0.8 1.1
84 93
93 79
Test Item 10.0 µg 100 87 14.1 61 76 14.5 0.9 0.9
89 77
72 90
Test Item 31.6 µg 69 78 13.3 101 86 14.1 0.8 1.1
71 84
93 73
Test Item 100 µg 67 68 1.7 69 81 12.5 0.7 1
67 80
70 94
Test Item 316 µg 83 80 7.4 68 85 17.6 0.8 1.1
86 83
72 103
Test Item 1000 µg 33 B 38 5.5 74 69 4.6 0.4 0.9
44 B 66
38 B 66
Test Item 2500 µg 9 B 18 9 43 B 52 8.6 0.2 0.7
17 B 60 B
27 B 54 B
Test Item 5000 µg 0 N 0 0 49 B 47 2.5 0 0.6
0 N 44 B
0 N 47 B
NaN3 10 µg 622 608 34.6 / / / 5.9 /
569 /
634 /
2-AA 2.5 µg / / / 798 1037 344.4 / 12.9
/ 882
/ 1432

TesterStrain:TA1535                                                                                    Experiment:2

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 16 19 3.6 12 10 1.5 1 1
23 10
18 9
Test Item 3.16 µg 20 18 2.5 10 12 3.2 0.9 1.2
15 16
18 11
Test Item 10.0 µg 20 20 2.5 9 9 2.5 1 0.9
17 7
22 12
Test Item 31.6 µg 23 19 4 12 11 2.3 1 1
15 8
18 12
Test Item 100 µg 19 21 2.6 12 10 2 1.1 1
20 10
24 8
Test Item 316 µg 18 19 2.3 7 8 2.3 1 0.8
22 7
18 11
Test Item 1000 µg 10 B 7 3.6 7 9 2 0.4 0.9
3 B 9
8 B 11
Test Item 2500 µg 1 B 2 1.5 10 9 1.2 0.1 0.8
2 B 8
4 B 8
Test Item 5000 µg 0 B 0 0 10 8 2.1 0 0.8
0 B 6
0 B 9
NaN3 10 µg 979 989 53.3 / / / 52.1 /
1047 /
942 /
2-AA 2.5 µg / / / 156 160 9.6 / 15.5
/ 171
/ 153

TesterStrain:TA1537                                                                                    Experiment:2

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 16 12 3.5 18 14 3.6 1 1
12 13
9 11
Test Item 3.16 µg 9 11 2.5 9 14 4.6 0.9 1
14 18
11 15
Test Item 10.0 µg 10 8 1.5 12 12 2 0.7 0.9
8 10
7 14
Test Item 31.6 µg 7 9 1.5 9 14 4.4 0.7 1
10 16
9 17
Test Item 100 µg 7 8 1.2 13 11 2 0.7 0.8
9 11
9 9
Test Item 316 µg 8 8 0.6 15 12 3.1 0.7 0.9
8 9
9 13
Test Item 1000 µg 8 8 0.6 8 10 2 0.6 0.7
7 10
8 12
Test Item 2500 µg 4 B 4 1.5 8 11 3.1 0.3 0.8
2 B 14
5 B 12
Test Item 5000 µg 1 B 2 1 10 10 2 0.2 0.7
3 B 12
2 B 8
4-NOPD 40 µg 76 78 11.7 / / / 6.4 /
91 /
68 /
2-AA 2.5 µg / / / 290 284 9 / 20.3
/ 274
/ 289

TesterStrain:TA102                                                                                      Experiment:2

Treatment Dose/plate REVERTANT COLONIES PER PLATE MUTATION FACTOR
Without activation (-S9) With activation (+S9)
Counts Mean SD Counts Mean SD -S9 +S9
A. dest. 　 311 276 30.8 335 316 16.8 1 1
253 310
264 303
Test Item 3.16 µg 302 299 9.5 340 330 36.5 1.1 1
288 290
306 361
Test Item 10.0 µg 249 262 18.4 343 345 25.1 0.9 1.1
254 321
283 371
Test Item 31.6 µg 255 266 11.5 320 305 16.6 1 1
265 307
278 287
Test Item 100 µg 286 276 9.3 322 325 4.6 1 1
273 322
268 330
Test Item 316 µg 242 248 7.4 304 300 4 0.9 0.9
245 296
256 299
Test Item 1000 µg 185 248 56.9 346 370 27 0.9 1.2
265 364
295 399
Test Item 2500 µg 234 257 21.7 316 280 31.7 0.9 0.9
277 256
260 268
Test Item 5000 µg 12 B 9 2.6 280 B 283 5.5 0 0.9
7 B 279 B
8 B 289 B
4-NOPD 1 µL 604 596 54.4 / / / 2.2 /
646 /
538 /
2-AA 10 µg / / / 812 929 105.4 / 2.9
/ 960
/ 1016

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative. The substance is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

A mutagenicity study with two experiments (used incorporation and pre-incubation method respectively) was performed on the test substance using S. typhimurium in the presence and absence of metabolic activation. Concentrations of 3.16, 10.0, 31.6 100, 316, 1000, 2500 and 5000 µg/plate, each was tested in triplicate. Cytotoxicity was observed from concentrations of 1000 μL/plate (with and without metabolic activation). All criteria of validity were met. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed at any concentration level, neither in the presence nor absence of metabolic activation in two experiments. The substance is considered to be non-mutagenic in this bacterial reverse mutation assay.