Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 February 2018 - 22 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254.
Test concentrations with justification for top dose:
- Experiment 1, dose range finding:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 5.4, 17, 52, 164, 512 and 1000 μg/plate
- Experiment 2:
TA1535, TA1537, TA98 and TA100 (without and with S9): 1.7, 5.4, 17, 52, 164 and 512 μg/plate
WP2uvrA (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: a solubility test was performed based on visual assessment. The test item formed a translucent suspension in DMSO. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test item concentrations were used within 2 hours after preparation.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: 2.5 μg/plate in DMSO for TA1537 (direct plate assay)
Remarks:
Without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
100 μL/plate DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA) in DMSO, 1 μg/plate for TA100 (direct plate assay) and for TA98, 2.5 μg/plate for TA1535 and TA1537, 5 μg/plate for TA100 (pre-incubation assay), 15 μg/plate for WP2uvrA
Remarks:
With S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 ± 2 minutes
- Exposure duration: 48 ± 4 hours

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: 10^9 bacteria per mL

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. The condition of the bacterial background lawn was evaluated, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
INTERPRETATION OF RESULTS
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (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 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.

ACCEPTABILITY CRITERIA
- The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test facility.
- The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
- No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Statistics:
Not performed.

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:
In the first experiment without and with S9, at 164 upwards and 512-1000 µg/plate, respectively. In the second experiment without and with S9, at 17 and upwards and at 164-512 µg/plate, respectively.
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:
In the first experiment without and with S9, at 164 and upwards and at 512 and 1000 μg/plate, respectively. In the second experiment without and with S9, at 17 and upwards and at 164 and 512 μg/plate, respectively.
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:
In the first experiment without and with S9, at 164 and upwards and at 512 and 1000 μg/plate, respectively. In the second experiment without and with S9, at 17 and upwards and at 164 μg/plate, respectively.
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:
In the first experiment without and with S9, at 164 μg/plate and upwards and at 512 μg/plate and upwards, respectively. In the second experiment without and with S9, at 5.4 and upwards and at 164 μg/plate, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Only in the second experiment without S9 at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Direct Plate Assay: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate and no precipitate was observed at the end of the incubation period.
Pre-Incubation Assay: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. Except in tester strain WP2uvrA in the presence of S9-mix where no toxicity was observed at any of the dose levels tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 – 1248 73 – 1206 55 – 1353 54 – 1051 365 – 1995 250 – 1977
Mean 846 219 787 353 1406 887
SD 146 119 345 162 258 349
n 2348 2229 2003 2234 2200 2276

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1848 408 - 2651 93 – 1951 111 - 1359
Mean 901 1232 1094 437
SD 168 343 477 149
n 2335 2327 2021 2085

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Oct 2015 and Oct 2017.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 - 27 3 – 20 3 – 23 8 - 41 8 - 55 63 – 176 54 - 160 10 – 59 9 - 69
Mean 10 11 6 7 16 23 108 107 25 32
SD 3 4 2 3 5 7 19 20 7 8
n 2356 2336 2264 2235 2319 2360 2341 2336 2075 2078

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Oct 2015 and Oct 2017.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- For both the direct plate assay and pre-incubation assay: cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Except in tester strain WP2uvrA in the presence of S9-mix where no toxicity was observed at any of the dose levels tested:
TA1535: In the first experiment without and with S9, at 164 and upwards and at 512 and 1000 μg/plate, respectively. In the second experiment without and with S9, at 17 and upwards and at 164 μg/plate, respectively.
TA1537: In the first experiment without and with S9, at 164 and upwards and at 512 and 1000 μg/plate, respectively. In the second experiment without and with S9, at 17 and upwards and at 164 and 512 μg/plate, respectively.
TA98: In the first experiment without and with S9, at 164 and upwards and at 512 and 1000 μg/plate, respectively. In the second experiment without and with S9, at 17 and upwards and at 164 μg/plate, respectively.
TA100: In the first experiment without and with S9, at 164 μg/plate and upwards and at 512 μg/plate and upwards, respectively. In the second experiment without and with S9, at 5.4 and upwards and at 164 μg/plate, respectively.
WP2uvrA: Only in the second experiment without S9 at 5000 μg/plate.

- In both assays no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles.

The test was performed in two independent experiments, first a direct plate assay and second a pre-incubation assay, in the absence and presence of S9-mix. Adequate negative and positive controls were included. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The substance precipitated on the plates only at the start of the test at 5000 μg/plate.

In the first mutation experiment, the substance was tested up to a concentration of 1000 μg/plate in the strains TA1535, TA1537 and TA98 (without and with S9). In the second mutation experiment, the substance was tested up to a concentration of 512 μg/plate in the strains TA1535, TA1537, TA98 and TA100 (with and without S9). The substance was tested up to 5000 μg/plate in the WP2uvrA strain (without and with S9). In both the first and second mutation experiment, no precipitation of the substance on the plates was observed. In both experiments, cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Except in tester strainWP2uvrA in the presence of S9-mix where no toxicity was observed at any of the dose levels tested. No biologically relevant increase in the number of revertants was observed in any of the tester strains, with and without S9. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.