Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May - 12 Jun 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254 (500 mg/kg bw); S9 mix was tested in two concentrations of 5% and 10% (v/v)
Test concentrations with justification for top dose:
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate for TA100 with (5% v/v) and without metabolic activation

The dose-range finding test is further reported as a part of Experiment 1.

Experiment 1: 52, 164, 512, 1600 and 5000 μg/plate for remaining strains with (5% v/v) and without metabolic activation
Experiment 2: 492, 878, 1568, 2800 and 5000 µg/plate for all strains with (10% v/v) and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: -S9, 0.75 µg/plate in DMSO for TA97; tert-butyl hydroperoxide (TBH): -S9, 250 µg/plate in DMSO for TA102; 2-aminoanthracene (2AA): +S9, 2.5, 5, 1, 1, 2 and 10 µg/plate in DMSO for strains TA1535, TA97, TA98, TA100, TA 100 and TA102, respectively
Remarks:
Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

DURATION : 48 ± 4 h

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, increase in the size of the microcolonies and the reduction of revertant colonies

OTHER:
Revertant colonies were counted with the Sorcerer Colony Counter.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than 2 times the concurrent control, and the total number of revertants in tester strains TA1535, and TA98 is not greater than 3 times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than 2 times the concurrent control, or the total number of revertants in tester strains TA1535 and, TA98 is greater than 3 times the concurrent control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in either the presence or absence of S9 mix.

HISTORICAL CONTROL DATA : The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Remarks on result:
other:
Remarks:
In strain TA102, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the presence of S9-mix at 512 μg/plate. Since the increase was less than two-fold (a maximum of 1.2-fold was reached), it was not considered to be biologically relevant.

Any other information on results incl. tables

Table 1. Results of dose-range finding test (as part of Experiment 1)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.)
  TA100
  -S9 mix +S9 mix (5%)
Positive control 706 ± 115 533 ± 63
Solvent control 91 ± 6 94 ± 27
1.7 104 ± 11 85 ± 12
5.4 105 ± 6 85 ± 6
17 95 ± 13 85 ± 19
52 86 ± 18 91 ± 16
164 97 ± 6 89 ± 7
512 106 ± 4 87 ± 9
1600 92 ± 6 95 ± 23
5000 90 ± 10 75 ± 13

Table 2. Results of Experiment 1

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.)
TA1535 TA98 TA102 TA97
  -S9 mix
Positive control 800 ± 5 1411 ± 56 876 ± 136 1605 ± 31
Solvent control 5 ± 2 10 ± 5 403 ± 24 136 ± 19
52 11 ± 4 11 ± 4 300 ± 11 131 ± 11
164 13 ± 2 14 ± 5 322 ± 26 122 ± 10
512 14 ± 3 14 ± 4 375 ± 23 121 ± 6
1600 7 ± 1 16 ± 4 388 ± 15 125 ± 8
5000 5 ± 4 10 ± 4 334 ± 21 113 ± 8
+S9 mix (5%)
Positive control 176 ± 18 1062 ± 76 732 ± 152 2074 ± 103
Solvent control 7 ± 4 17 ± 2 371 ± 21 142 ± 12
52 9 ± 4 18 ± 5 420 ± 27 154 ± 21
164 7 ± 3 17 ± 1 394 ± 71 155 ± 7
512 8 ± 4 17 ± 2 440 ± 36 139 ± 13
1600 9 ± 3 17 ± 8 408 ± 27 147 ± 29
5000 4 ± 1 13 ± 4 401 ± 16 137 ± 20

 

Table 3. Results of Experiment 2

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.)
TA1535 TA98 TA100 TA102 TA97
  -S9 mix
Positive control 922 ± 27 1172 ± 232 1266 ± 119 1331 ± 296 1488 ± 76
Solvent control 10 ± 5 14 ± 6 104 ± 11 327 ± 41 152 ± 6
492 14 ± 1 30 ± 9 109 ± 13 238 ± 51 152 ± 9
878 10 ± 5 24 ± 5 102 ± 25 267 ± 31 163 ± 20
1568 10 ± 3 12 ± 4 105 ± 8 306 ± 29 155 ± 7
2800 14 ± 6 10 ± 2 122 ± 3 239 ± 46 155 ± 15
5000 10 ± 2 18 ± 9 104 ± 11 247 ± 18 171 ± 21
  +S9 mix (10%)
Positive control 251 ± 71 789 ± 88 1206 ± 152 718 ± 53 1812 ± 179
Solvent control 21 ± 6 24 ± 5 92 ± 23 315 ± 52 174 ± 20
492 17 ± 3 26 ± 9 90 ± 10 313 ± 19 177 ± 6
878 13 ± 2 18 ± 5 85 ± 15 303 ± 30 197 ± 24
1568 12 ± 4 21 ± 3 80 ± 6 326 ± 34 173 ± 7
2800 14 ± 2 22 ± 4 79 ± 13 300 ± 11 183 ± 5
5000 8 ± 4 21 ± 4 87 ± 1 308 ± 29 167 ± 20

Table 4. Historical solvent control data from experiments performed between May 2015 and May 2017

  TA1535 TA97 TA98 TA100 TA102
S9-mix - + - + - + - + - +
Range 3 -36 3 - 32 85 - 191 93 - 199 8 - 41 9 - 55 66 - 161 63 - 160 195 - 475 264 - 428
Mean 11 11 122 151 16 23 105 105 269 336
SD 4 4 31 27 5 7 19 20 44 37
n 2057 2039 45 45 2023 2083 2027 2033 267 288

SD = standard deviation

n = number ob observations

Table 5. Historical positive control data from esperiments performed between May 2015 and May 2017

  TA1535 TA97 TA98 TA100 TA102
S9-mix - + - + - + - + - +
Range 125 - 1381 78 - 1058 652 - 2026 915 – 2405 410 – 1995 250 - 1977 537 – 1848 408 - 2651 618 – 1215 628 – 1154
Mean 839 220 1131 1785 1369 929 908 1330 857 885
SD 153 112 312 446 310 345 178 324 255 153
n 2065 1967 48 45 1920 2014 2007 2020 10 26

SD = standard deviation

n = number ob observations

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted study the substance was not mutagenic in any of the five Salmonella typhimurium strains (TA1535, TA97, TA98, TA100 and TA102) tested with and without metabolic activation.