Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct - 20 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-phenylimidazole
EC Number:
211-581-7
EC Name:
2-phenylimidazole
Cas Number:
670-96-2
Molecular formula:
C9H8N2
IUPAC Name:
2-phenyl-1H-imidazole
Test material form:
solid: flakes

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 rats treated with phenobarbital and β-naphthoflavone.
Test concentrations with justification for top dose:
Preliminary toxicity study: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation for tester strains TA98 and TA100

Based on the results of the preliminary toxicity study the following concentration levels were used for all tester strains in the main experiment:
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine: -S9: 10 and 40 µg/plate for TA98 and TA1537, respectively; 2-aminoanthracene: +S9: 2.5 or 10 µg/plate for all strains
Remarks:
The biological activity of the metabolic activation system was investigated in a quality control using 2-aminoanthracene and benzo[a]pyrene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation, Experiment I); preincubation (Experiment II)

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

NUMBER OF REPLICATIONS: Triplicates


DETERMINATION OF CYTOTOXICITY
- Method: Clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number if revertants occurs and/or
- a biologically relevant positive response for at least one of the dose group occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to the OECD guideline, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with (Experiment II) and without metabolic activation (Experiment I and II)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with (Experiment II) and without metabolic activation (Experiment I and II)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without metabolic activation in both Experiments (I and II).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with (Experiment II) and without metabolic activation (Experiment I and II)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate without metabolic activation (Experiment I and II)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in Experiment I and II with and without metabolic activation, respectively.

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed in tester strains TA98 and TA100. The following concentration levels were tested: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation. The highest concentration level of 5000 µg/plate exhibited slight toxicity and was therefore chosen for the main experiment.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data, therefore the test was considered to be valid.
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data, therefore the test was considered to be valid.

Any other information on results incl. tables

Table 2: Summary of results (Experiment I)

  TA98 TA100 TA1535 TA1537 TA102
  without metabolic activation with metabolic activation without metabolic activation with metabolic activation without metabolic activation with metabolic activation without metabolic activation with metabolic activation without metabolic activation with metabolic activation
  Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor
Distilled water 22 5.7 0.9 27 7.8 1.1 112 7.5 1.5 73 11.8 1.0 16 2.5 1.0 13 2.9 0.8 29 0.6 1.0 20 2.1 1.0 293 0.6 1.1 313 36.1 1.1
DMSO 25 2.6 1.0 24 5.2 1.0 76 7.4 1.0 70 8.7 1.0 16 3.5 1.0 16 2.1 1.0 30 1.2 1.0 21 3.5 1.0 269 1.2 1.0 294 12.9 1.0
31.6 µg/plate 23 7.6 0.9 27 1.2 1.1 91 11.2 1.2 73 13.5 1.0 12 2.6 0.8 14 3.5 0.9 24 2.1 0.8 24 1.7 1.1 283 2.1 1.1 294 21.0 1.0
100 µg/plate 23 6.1 1.0 20 5.3 0.8 83 6.9 1.1 69 11.6 1.0 16 2.1 1.0 13 6.2 0.8 22 1.0 0.7 21 0.6 1.0 259 1.0 1.0 315 7.4 1.1
316 µg/plate 26 13.5 1.1 24 1.5 1.0 68 16.2 0.9 94 26.1 1.3 14 2.9 0.9 12 1.5 0.8 23 1.5 0.8 21 1.5 1.0 266 1.5 1.0 301 28.1 1.0
1000 µg/plate 26 5.0 0.7 25 6.0 1.0 68 4.0 0.9 88 22.9 1.3 14 3.5 0.9 10 0.0 0.6 20 1.5 0.7 20 4.0 0.9 244 1.5 0.9 276 2.1 0.9
2500 µg/plate 17 2.0 0.3 18 2.5 0.7 47 9.6 0.6 59 10.3 0.8 13 2.3 0.8 12 4.0 0.7 24 1.0 0.8 22 2.1 1.0 201 1.0 0.7 237 5.0 0.8
5000 µg/plate 6 3.5 0.5 12 6.1 0.5 15 3.5 0.2 47 23.8 0.7 13 2.5 0.8 5 4.7 0.3 15 7.0 0.5 22 2.6 1.0 138 7.0 0.5 192 22.3 0.7
4-NOPD 471 20.1 18.8 - - - - - - - - - - - - - - - 111 13.2 3.7 - - - - - - - - -
NA - - - - - - 736 157.8 9.7 - - - 1176 61.7 73.5 - - - - - - - - - - - - - - -
MMS - - - - - - - - - - - - - - - - - - - - - - - - 977 30.1 3.6 - - -
2-AA - - - 2073 307.5 86.4 - - - 1522 53.2 21.7 - - - 249 32.0 15.9 - - - 108 14.0 5.1 - - - 806 8.5 1.7

4-NOPD: 4-nitro-o-phenylene-diamine

NA: sodium azide

MMS: methylmethanesulphonate

2-AA: 2-Aminoanthracene

Table 2: Summary of results (Experiment II)

  TA98 TA100 TA1535 TA1537 TA102
  without metabolic activation   with metabolic activation without metabolic activation with metabolic activation without metabolic activation with metabolic activation without metabolic activation with metabolic activation without metabolic activation with metabolic activation
  Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor Mean SD Mutation factor
Distilled water 26 5.0 1.0 26 5.3 1.1 98 4.2 1.1 87 7.5 1.1 22 0.6 1.0 15 3.2 0.9 17 1.0 1.0 20 1.5 1.0 275 37.8 1.0 370 53.1 1.2
DMSO 25 7.2 1.0 23 5.2 1.0 92 21.1 1.0 77 8.5 1.0 23 3.8 1.0 16 1.5 1.0 17 2.0 1.0 20 2.1 1.0 275 23.1 1.0 302 35.0 1.0
10.0 µg/plate 27 5.8 1.1 29 5.1 1.3 99 7.0 1.1 86 20.6 1.1 26 2.0 1.1 14 1.7 0.9 17 1.5 1.0 20 2.6 1.0 287 3.5 1.0 300 10.6 1.0
31.6 µg/plate 21 2.6 0.8 31 3.0 1.3 102 6.1 1.1 83 1.2 1.1 22 2.1 1.0 17 6.2 1.0 17 1.5 1.0 22 2.1 1.1 277 12.7 1.0 299 31.6 1.0
100 µg/plate 21 2.6 0.8 35 4.6 1.5 100 16.8 1.1 78 1.5 1.0 22 4.4 0.9 16 4.0 1.0 19 1.5 1.1 22 1.5 1.1 300 9.0 1.1 364 10.4 1.2
316 µg/plate 22 0.6 0.9 31 0.6 1.3 88 2.6 1.0 77 2.1 1.0 22 3.8 0.9 19 4.4 1.2 19 2.1 1.1 25 2.1 1.3 265 21.2 1.0 309 23.0 1.0
1000 µg/plate 22 7.5 0.9 28 6.0 1.2 84 9.3 0.9 85 6.8 1.1 27 3.1 1.1 18 5.2 1.1 18 1.5 1.1 21 3.2 1.1 277 36.2 1.0 357 29.0 1.2
2500 µg/plate 21 5.7 0.9 26 4.0 1.1 54 2.9 0.6 83 9.1 1.1 22 2.0 0.9 13 1.7 0.8 15 3.0 0.9 22 1.0 1.1 270 31.0 1.0 357 24.1 1.2
5000 µg/plate 11 4.9 0.4 7 4.0 0.3 18 16.3 0.2 26 7.9 0.3 6 1.2 0.2 4 1.5 0.2 4 1.5 0.2 8 1.5 0.4 133 17.2 0.5 182 82.2 0.6
4-NOPD 295 23.1 11.8 - - - - - - - - - - - - - - - 96 19.3 5.6 - - - - - - - - -
NA - - - - - - 634 44.7 6.9 - - - 836 107.0 35.8 - - - - - - - - - - - - - - -
MMS - - - - - - - - - - - - - - - - - - - - - - - - 692 41.2 2.5 - - -
2-AA - - - 1693 477.0 73.6 - - - 1136 202.0 14.8 - - - 97 14.8 5.9 - - - 127 14.0 6.5 - - - 944 95.2 3.1

4-NOPD: 4-nitro-o-phenylene-diamine

NA: sodium azide

MMS: methylmethanesulphonate

2-AA: 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative