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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 2008
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Range-finding test:
5 µL/plate with and without metabolic activation
Experiment 1:
0.05, 0.15, 0.5, 1,5 and 5 µL/plate with and without metabolic activation
Experiment 2a:
0.08, 0.16, 0.31, 0.63, 1.25, 2.5 and 5 µL/plate with and without metabolic activation
Experiment 2b:
0.3, 0.6, 1.1, 2.2, 4.4, 6.6 and 8.8 µL/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: demineralized water
- Justification for choice of solvent/vehicle: Demineralized water was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-nitro-1,2-phenylene diamine: 20 µg/plate for TA97a, TA98 and TA102 without S9 mix; 2-aminoanthracene: 1 µg/plate for TA97a, TA100, TA102 and TA1535 with S9 mix
Remarks:
Each batch of S9 is characterized with a mutagen that requires metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, range-finding study, Experiment 1); preincubation (Experiment 2a and 2b)

DURATION
- Preincubation period: 20 min (Experiment 2a and 2b)
- Exposure duration: 48 h (Experiment 1, 2a and 2b)

NUMBER OF REPLICATIONS: triplicates each in three independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn; number of revertants
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: S. typhimurium TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item showed no precipitates on the plates at all tested concentrations in all experiments.

RANGE-FINDING/SCREENING STUDIES: No toxicity of the test item was observed at the limit concentration of 5 µL/plate in all tester strains with and without metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data: All positive controls showed mutagenic effects with and without metabolic activation and were within the historical control data ranges (please refer to "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: All determined values (Experiment 1 and 2a) and nearly all (Experiment 2b) determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (please refer to "any other information on results incl. tables"). Bacteria strains are living biological systems, therefore variations in behaviour are not unusual. Each bacteria strain in this study has its own characteristic spontaneous revertant colony number. Day to day variations in the number of spontaneous revertant colonies are usual. The variations of the values of all bacteria strains lie in an acceptable range. Evaluation of the mutagenicity of the test item and validity of the study was not affected by these variations; this is why no critical impact on the outcome of the study is expected.

Any other information on results incl. tables

Table 1: Mean number of revertants Experiment 1

Strain   TA97a TA98 TA100 TA102 TA1535
Metabolic activation   -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Demin. water Mean 73 81 26 25 77 93 308 353 19 23
sd 7.6 11.3 4.7 6.2 7.5 7.0 45.1 4.6 1.0 4.2
DMSO Mean 67 85 26 29 72 83 305 292 19 21
sd 3.6 13.0 3.6 6.5 7.8 9.5 45.5 56.7 3.8 4.7
Positive controls Mean 520 609 407 86 324 1001 1005 1363 224 247
sd 84.3 136.8 56.0 6.8 55.6 0.0 334.9 224.2 49.2 48.1
f(I) 7.76 7.16 15.65 2.97 4.21 12.06 3.30 4.67 11.79 11.76
5 µL/plate Mean 68 72 29 30 75 70 304 376 20 19
sd 3.5 16.3 3.5 9.0 9.0 13.6 76.6 8.0 2.5 1.7
f(I) 0.93 0.89 1.12 1.20 0.97 0.75 0.99 1.07 1.05 0.83
1.5 µL/plate Mean 73 64 27 33 76 81 340 367 17 21
sd 9.6 5.5 7.2 7.0 12.4 7.6 56.7 26.0 2.5 3.6
f(I) 1.00 0.79 1.04 1.32 0.99 0.87 1.10 1.04 0.89 0.91
0.5 µL/plate Mean 71 70 24 24 83 80 368 351 20 15
sd 4.2 12.1 4.6 6.1 9.1 11.5 30.2 12.2 2.1 3.2
f(I) 0.97 0.86 0.92 0.96 1.08 0.86 1.19 0.99 1.05 0.65
0.15 µL/plate Mean 75 78 24 28 82 92 353 307 22 21
sd 5.0 19.7 5.5 7.0 8.2 14.4 30.3 24.4 2.6 2.1
f(I) 1.03 0.96 0.92 1.12 1.06 0.99 1.15 0.87 1.16 0.91
0.05 µL/plate Mean 82 79 31 27 81 73 333 289 20 18
sd 17.4 10.8 0.0 4.7 15.1 9.6 33.5 34.0 2.6 4.0
f(I) 1.12 0.98 1.19 1.08 1.05 0.78 1.08 0.82 1.05 0.78

Table 2: Mean number of revertants Experiment 2a

Strain   TA97a TA98 TA100 TA102 TA1535
Metabolic activation    -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Demin. water Mean 89 106 32 30 104 103 267 317 26 24
sd 13.1 6.8 14.2 5.5 25.4 24.8 40.3 30.0 4.6 6.5
DMSO Mean 85 91 26 30 92 113 301 307 21 24
sd 5.5 28.9 8.1 5.1 8.0 22.9 40.3 62.3 0.6 2.1
Positive controls Mean 599 671 212 165 339 1001 1331 1485 223 228
sd 176.4 119.5 33.0 8.3 24.1 0.0 23.1 28.1 20.1 16.0
f(I) 7.05 7.37 8.15 5.50 3.26 8.86 4.42 4.84 8.58 9.50
5 µL/plate Mean 81 97 25 28 99 141 209 232 25 26
sd 1.2 6.4 1.5 7.8 1.2 18.1 62.8 52.0 2.3 5.3
f(I) 0.91 0.92 0.78 0.93 0.95 1.37 0.78 0.73 0.96 1.08
2.5 µL/plate Mean 87 100 30 30 121 115 260 303 19 25
sd 17.3 27.0 3.5 2.3 8.1 1.2 21.2 22.7 2.5 5.5
f(I) 0.98 0.94 0.94 1.00 1.16 1.12 0.97 0.96 0.73 1.04
1.25 µL/plate Mean 86 92 28 34 104 97 275 327 26 24
sd 13.1 16.1 0.6 2.0 18.5 12.1 31.1 50.6 4.0 2.0
f(I) 0.97 0.87 0.88 1.13 1.00 0.94 1.03 1.03 1.00 1.00
0.63 µL/plate Mean 72 66 23 34 106 114 252 265 20 24
sd 21.5 12.4 2.6 1.5 15.6 18.4 4.0 82.0 0.6 2.5
f(I) 0.81 0.62 0.72 1.13 1.02 1.11 0.94 0.84 0.77 1.00
0.31 µL/plate Mean 85 104 29 33 85 113 251 311 21 21
sd 8.7 22.3 8.6 1.7 16.0 13.3 30.0 34.0 2.6 4.5
f(I) 0.96 0.98 0.91 1.10 0.82 1.10 0.94 0.98 0.81 0.88
0.16 µL/plate Mean 87 95 31 34 96 89 280 275 23 24
sd 20.4 16.3 2.5 1.7 14.3 17.1 16.0 53.7 6.2 4.9
f(I) 0.98 0.90 0.97 1.13 0.92 0.86 1.05 0.87 0.88 1.00
0.08 µL/plate Mean 79 90 34 34 90 107 245 285 25 27
sd 17.5 13.1 2.1 5.9 17.6 26.9 31.1 26.6 8.5 2.9
f(I) 0.89 0.85 1.06 1.13 0.87 1.04 0.92 0.90 0.96 1.13

Table 3: Mean number of revertants Experiment 2b

Strain   TA97a TA98 TA100 TA102 TA1535
Metabolic activation   -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Demin. water Mean 82 120 43 42 147 140 288 287 36 40
sd 8.2 36.5 3.6 4.7 4.7 9.0 34.2 32.3 5.7 2.3
DMSO Mean 108 118 40 37 138 139 261 281 35 37
sd 12.2 25.7 1.2 1.5 2.5 1.5 32.1 34.9 4.0 3.6
Positive controls Mean 656 533 461 177 792 1001 1272 1288 208 165
sd 72.3 34.0 53.3 57.9 27.7 0.0 162.4 367.4 10.6 15.1
f(I) 6.07 4.52 11.53 4.78 5.39 7.20 4.87 4.58 5.78 4.46
8.8 µL/plate Mean 89 154 38 39 124 139 215 273 34 31
sd 6.7 2.3 2.9 7.6 14.8 24.1 28.1 40.1 5.6 0.6
f(I) 1.09 1.28 0.88 0.93 0.84 0.99 0.75 0.95 0.94 0.78
6.6 µL/plate Mean 118 127 40 44 145 144 265 271 32 31
sd 33.7 16.2 2.1 6.7 9.6 6.0 36.3 30.3 2.6 4.0
f(I) 1.44 1.06 0.93 1.05 0.99 1.03 0.92 0.94 0.89 0.78
4.4 µL/plate Mean 94 120 38 35 150 125 253 239 35 33
sd 31.1 38.7 2.9 3.2 16.2 36.8 37.2 16.2 5.6 4.9
f(I) 1.15 1.00 0.88 0.83 1.02 0.89 0.88 0.83 0.97 0.83
2.2 µL/plate Mean 102 95 36 41 171 119 247 200 39 30
sd 24.8 11.5 2.3 3.5 8.4 41.1 87.3 36.0 2.5 7.0
f(I) 1.24 0.79 0.84 0.98 1.16 0.85 0.86 0.70 1.08 0.75
1.1 µL/plate Mean 130 135 38 33 131 132 216 233 40 29
sd 2.1 7.5 7.4 1.5 2.3 2.6 42.3 12.9 2.1 7.2
f(I) 1.59 1.13 0.88 0.79 0.89 0.94 0.75 0.81 1.11 0.73
0.6 µL/plate Mean 132 117 41 37 139 133 245 321 37 36
sd 11.1 18.6 5.1 6.1 15.7 4.2 28.9 33.3 1.5 6.2
f(I) 1.61 0.98 0.95 0.88 0.95 0.95 0.85 1.12 1.03 0.90
0.3 µL/plate Mean 125 121 42 39 145 135 241 260 39 42
sd 17.0 6.1 4.2 4.9 18.2 9.7 47.4 14.4 4.0 3.5
f(I) 1.52 1.01 0.98 0.93 0.99 0.96 0.84 0.91 1.08 1.05

Applicant's summary and conclusion

Conclusions:
The test substance is not mutagenic in the Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.