Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jul - 03 Aug 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 2020
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
adopted in 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales, Düsseldorf, Germany
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 TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254.
Prior to first use, each batch was checked for its metabolising capacity by using reference mutagens; appropriate activity was demonstrated.
Test concentrations with justification for top dose:
First and second experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation
Dose levels were routinely determined on the basis of a standard protocol: If not limited by solubility, 5000 µg/plate or 5 µL/plate were used as highest dose. The results of the first experiment were considered as a pre-test for toxicity. Concentrations of the second experiment were chosed based on the results of the first experiment.
Vehicle / solvent:
Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
mitomycin C
other: nitrofurantoin, -S9, 0.2 and 0.4 µg/plate in DMSO for TA100; 4-nitro-1,2-phenylene diamine, -S9, 10 and 20 µg/plate in DMSO for TA1537, 0.5 and 1 µg/plate in DMSO for TA98; 2-aminoanthracene, +S9, 3 and 6 µg/plate in DMSO for all strains
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments : 2

METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1) and preincubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytotoxicity was determined as background growth inhibition, dose-dependent reduction in mutant count per plate and by titer determination.

Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100 and TA98 this increase should be about twice that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
evident as a reduction in titer at ≥ 1581 µg/plate for all strains in the first experiment (plate incorporation test) and for strain TA 100 at 5000 µg/plate in the second experiment (pre-incubation test).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS :
- Precipitation and time of the determination: There was no precipitation at any test item concentration in any experiment, neither with nor without S9 mix.

CYTOTOXICITY:
Cytotoxicity, evident as a reduction in titer was noted for all strains in the first experiment at ≥ 1581 µg/plate (plate incorporation test) and for strain TA 100 at 5000 µg/plate in the second experiment (pre-incubation test). There was no inhibition of the bacterial background lawn and no decrease in the number of spontaneous revertant colonies.

HISTORICAL CONTROL DATA: please refer to document "Historical control data generated in the testing laboratory from 1999 to 2008" attached.

Any other information on results incl. tables

Table 1: Mean number of revertant colonies per plate observed in the first experiment (plate incorporation test)

Concentration
(µg/plate)
TA 1535 TA 100 TA 1537 TA 98 TA 102
Metabolic activation (-S9) (+S9) (-S9) (+S9) (-S9) (+S9) (-S9) (+S9) (-S9) (+S9)
DMSO 8 ± 1 11 ± 2 110 ± 15 177 ± 22 6 ± 2 8 ± 1 23 ± 2 39 ± 2 294 ± 5 313 ± 64
16 7 ± 1 8 ± 1 101 ± 19 149 ± 23 6 ± 2 7 ± 1 22 ± 6 44 ± 7 301 ± 34 326 ± 76
50 8 ± 2 9 ± 1 96 ± 14 159 ± 29 5 ± 2 8 ± 0 22 ± 4 44 ± 2 269 ± 7 389 ± 28
158 8 ± 2 9 ± 2 102 ± 3 144 ± 13 5 ± 1 7 ± 2 21 ± 5 46 ± 9 279 ± 8 280 ± 90
500 7 ± 2 8 ± 2 115 ± 2 165 ± 21 4 ± 1 8 ± 1 18 ± 3 40 ± 4 205 ± 3 299 ± 51
1581 7 ± 1 8 ± 2 96 ± 15 114 ± 17 4 ± 1 8 ± 1 22 ± 5 43 ± 7 195 ± 60 333 ± 71
5000 8 ± 4 7 ± 2 98 ± 16 91 ± 11 5 ± 2 6 ± 1 21 ± 9 33 ± 5 206 ± 65 259 ± 78
Positive control# NaN3 2-AA NF 2-AA 4-NPDA 2-AA 4-NPDA 2-AA MMC 2-AA
a) 772 ± 28 113 ± 8 300 ± 59 1298 ± 138 35 ± 3 193 ± 38 109 ± 7 1731 ± 324 846 ± 18 582 ± 18
b) 917 ± 24 82 ± 2 398 ± 27 1396 ± 258 36 ± 8 48 ± 6 132 ± 6 1821 ± 210 1003 ± 117 1269 ± 170
#: NaN3: Sodium azide, NF: Nitrofurantoin, 4-NPDA: 4-Nitro-1,2-phenylene diamine, MMC: Mitomycin C, 2-AA: 2-Aminoanthracene
a) lower concentration, 10 µg/plate for NaN3, 0.2 µg/plate for NF, 10µg/plate for 4-NDPA for TA1537, 0.5 µg/plate for TA98, 0.2 µg/plate for MMC, 3 µg/platefor 2-AA
b) upper concentration, 20 µg/plate for NaN3, 0.4 µg/plate for NF, 20µg/plate for 4-NDPA for TA1537, 1 µg/plate for TA98, 0.4 µg/plate for MMC, 6 µg/plate for 2-AA

Table 2: Mean number of revertant colonies per plate observed in the second experiment (pre-incubation test)

Concentration
(µg/plate)
TA 1535 TA 100 TA 1537 TA 98 plate
Metabolic activation (-S9) (+S9) (-S9) (+S9) (-S9) (+S9) (-S9) (+S9) (-S9) (+S9)
DMSO 9 ± 3 13 ± 3 134 ± 24 177 ± 21 7 ± 1 10 ± 3 16 ± 1 30 ± 4 249 ± 20 316 ± 11
16 9 ± 2 14 ± 3 146 ± 3 190 ± 20 7 ± 1 10 ± 4 17 ± 3 27 ± 4 251 ± 14 303 ± 15
50 8 ± 2 13 ± 2 131 ± 15 191 ± 7 7 ± 1 8 ± 1 17 ± 6 34 ± 3 251 ± 19 277 ± 47
158 11 ± 3 13 ± 2 146 ± 27 218 ± 21 7 ± 1 8 ± 1 18 ± 5 32 ± 2 216 ± 15 335 ± 18
500 8 ± 1 11 ± 4 122 ± 11 214 ± 31 6 ± 1 7 ± 1 20 ± 2 27 ± 4 250 ± 8 342 ± 27
1581 7 ± 2 8 ± 1 153 ± 5 188 ± 3 7 ± 2 8 ± 3 18 ± 1 31 ± 4 239 ± 9 335 ± 13
5000 8 ± 1 11 ± 2 132 ± 10 173 ± 10 7 ± 1 7 ± 1 19 ± 3 26 ± 6 254 ± 29 277 ± 36
Positive control# NaN3 2-AA NF 2-AA 4-NPDA 2-AA 4-NPDA 2-AA Cumene 2-AA
a) 1074 ± 15 112 ± 13 503 ± 48 2459 ± 451 34 ± 2 273 ± 36 72 ± 6 1538 ± 94 447 ± 8 539 ± 39
b) 850 ± 13 68 ± 21 927 ± 23 2553 ± 325 50 ± 4 183 ± 12 114 ± 17 2019 ± 117 454 ± 29 874 ± 35
#: NaN3: Sodium azide, NF: Nitrofurantoin, 4-NPDA: 4-Nitro-1,2-phenylene diamine, 2-AA: 2-Aminoanthracene
a) lower concentration, 10 µg/plate for NaN3, 0.2 µg/plate for NF, 10µg/plate for 4-NDPA for TA1537, 0.5 µg/plate for TA98, 50 µg/plate for Cumene, 3 µg/platefor 2-AA
b) upper concentration, 20 µg/platefor NaN3, 0.4 µg/plate for NF, 20µg/plate for 4-NDPA for TA1537, 1 µg/plate for TA98, 75µg/plate for Cumene, 6 µg/plate for 2-AA

Applicant's summary and conclusion

Conclusions:
The test item was tested for bacterial mutagenicity in the Ames test according to OECD guideline 471. Under the conditions of the test, the test item was not mutagenic in S. typhimuirum strains TA98, TA100, TA102, TA1535 and TA1537 with and without metabolic activation.