Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Apr - 10 May 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine: -S9: 10 and 50 µg/plate for TA98 and TA1537, respectively; 2-aminoanthracene: +S9: 2.5 or 10 µg/plate for all strains
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation; Experiment I); preincubation (Experiment II)

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

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

- OTHER: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to and including the highest dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I, as all strains were tested in the pre-experiment and the results were valid.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control data fell within the range of the historical control data (please refer to Table 3 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control data (solvent and untreated) fell within the range of the historical control data (please refer to Table 3 under "any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Summary of Experiment I (plate incorporation)

Metabolic Activation Test Group Dose Level per group and per plate Revertant Colony Counts (Mean of 3 plates ±SD)
      TA 1535 TA 1537 TA 98 TA 100 WP2uvrA
Without Activation Deionised water   12 ± 3 8 ± 3 30 ± 10 195 ± 8 40 ± 3
Untreated   9 ± 2 9 ± 4 24 ± 7 195 ± 7 32 ± 8
Test substance 3 µg 10 ± 3 10 ± 4 23 ± 3 191 ± 9 33 ± 6
10 µg 13 ± 6 9 ± 2 25 ± 8 190 ± 22 39 ± 4
33 µg 10 ± 3 11 ± 5 27 ± 3 188 ± 15 44 ± 5
100 µg 13 ± 4 7 ± 3 29 ± 10 191 ± 17 38 ± 4
333 µg 12 ± 4 7 ± 2 24 ± 5 198 ± 23 40 ± 3
1000 µg 8 ± 3 9 ± 3 31 ± 6 209 ± 16 43 ± 5
2500 µg 10 ± 3 10 ± 5 30 ± 5 196 ± 14 44 ± 4
5000 µg 11 ± 3 9 ± 2 30 ± 6 182 ± 16 43 ± 7
NaN3 10 µg 1327 ± 26     2265 ± 83  
4-NOPD 10 µg     354 ± 18    
4-NOPD 50 µg   69 ± 11      
MMS 2.0 µL         922 ± 16
With Activation Deionised water   12 ± 3 17 ± 4 33 ± 3 165 ± 12 43 ± 5
Untreated   12 ± 4 13 ± 2 46 ± 4 163 ± 4 52 ± 5
Test substance 3 µg 15 ± 2 16 ± 2 37 ± 6 166 ± 26 47 ± 3
10 µg 14 ± 3 18 ± 3 43 ± 5 171 ± 29 50 ± 4
33 µg 11 ± 5 13 ± 3 32 ± 6 189 ± 10 44 ± 3
100 µg 14 ± 5 12 ± 3 46 ± 4 175 ± 4 47 ± 10
333 µg 12 ± 3 13 ± 4 42 ± 4 154 ± 16 39 ± 9
1000 µg 11 ± 4 11 ± 1 37 ± 6 163 ± 8 45 ± 3
2500 µg 8 ± 1 9 ± 3 29 ± 8 168 ± 11 50 ± 4
5000 µg 11 ± 1 10 ± 5 33 ± 3 175 ± 11 50 ± 3
2-AA 2.5 µg 475 ± 27 169 ± 28 3890 ± 419 4361 ± 172  
2-AA 10.0 µg         358 ± 35

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methanesulfonate

2-AA = 2-aminoanthracene

Table 2: Summary of Experiment II (pre-incubation)

Metabolic Activation Test Group Dose Level per group and per plate Revertant Colony Counts (Mean of 3 plates ±SD)
      TA 1535 TA 1537 TA 98 TA 100 WP2uvrA
Without Activation Deionised water   11 ± 3 10 ± 1 25 ± 5 173 ± 12 34 ± 3
Untreated   11 ± 2 9 ± 3 25 ± 4 179 ± 8 39 ± 7
Test substance 33 µg 10 ± 1 10 ± 5 27 ± 6 190 ± 6 37 ± 5
100 µg 15 ± 4 8 ± 2 23 ± 8 182 ± 28 33 ± 3
333 µg 14 ± 6 7 ± 3 26 ± 6 196 ± 23 46 ± 1
1000 µg 14 ± 2 12 ± 3 25 ± 3 172 ± 14 35 ± 7
2500 µg 13 ± 2 7 ± 0 33 ± 6 162 ± 19 35 ± 1
5000 µg 8 ± 2 9 ± 1 28 ± 8 184 ± 7 47 ± 3
NaN3 10 µg 1095 ± 33     1643 ± 143  
4-NOPD 10 µg     349 ± 5    
4-NOPD 50 µg   89 ± 17      
MMS 2.0 µL         797 ± 128
With Activation Deionised water   10 ± 1 13 ± 3 39 ± 12 179 ± 13 55 ± 12
Untreated   13 ± 6 17 ± 5 43 ± 10 137 ± 18 47 ± 11
Test substance 33 µg 12 ± 2 14 ± 5 42 ± 13 150 ± 13 51 ± 5
100 µg 11 ± 5 11 ± 5 35 ± 6 163 ± 13 54 ± 5
333 µg 9 ± 1 15 ± 3 29 ± 4 172 ± 6 55 ± 12
1000 µg 16 ± 8 12 ± 4 32 ± 6 144 ± 13 55 ± 7
2500 µg 15 ± 4 15 ± 1 31 ± 6 157 ± 18 55 ± 6
5000 µg 15 ± 6 14 ± 5 34 ± 4 141 ± 11 40 ± 10
2-AA 2.5 µg 301 ± 5 126 ± 10 2920 ± 93 1842 ± 65  
2-AA 10.0 µg         329 ± 58

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methanesulfonate

2-AA = 2-aminoanthracene

Table 3: Historical Data

Strain   without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
TA 1535 Solvent control 12 2.5 6 25 12 2.5 7 26
Untreated control 12 3.1 6 28 12 2.9 7 26
Positive control 1130 143.1 334 1816 388 58.2 176 668
TA1537 Solvent control 10 2.2 6 19 13 3.5 7 30
Untreated control 11 2.7 5 21 14 4.0 7 31
Positive control 82 12.7 43 157 191 60.8 83 434
TA 98 Solvent control 25 4.4 13 43 34 6.2 15 58
Untreated control 27 4.9 12 43 37 6.5 11 57
Positive control 378 73.7 211 627 3949 771.8 360 6586
TA 100 Solvent control 156 26.0 78 209 148 32.3 73 208
Untreated control 176 23.6 79 217 172 25.4 85 218
Positive control 1966 293.2 498 2767 3798 830.4 536 6076
WP2uvrA Solvent control 41 5.6 27 63 50 6.8 28 72
Untreated control 42 5.8 30 63 52 6.8 36 88
Positive control 798 362.7 319 4732 378 112.6 167 1265

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the source substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.