Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 2017 - 30 March 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:
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 (incl. certificate)
Remarks:
d.d. 3 November 2015
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Name as cited in report: X-19924
Appearance: thick amber paste
Storage: at room temperature
Specific details on test material used for the study:
Purity correction factor: 1.138
Stability at higher temperatures: stable at a maximum temperature of 60°C for a maximum duration of 30 minutes

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
Preliminary test (without and with 5% S9-mix) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study:
TA1535, TA1537 and TA98: without and with 5% S9-mix: 0.54, 1.7, 5.4, 17, 52, 164 and 512 µg/plate

Experiment 2:
TA1535, TA1537, TA98 and TA100: without and with 10% S9-mix: 12.5, 25, 50, 100, 200 and 400 µg/plate
WP2uvrA: without and with 10% S9-mix: 400, 878, 1568, 2800 and 5000 µg/plate
Additional:
TA1537: without S9-mix: 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/plate
WP2uvrA: with 10% S9-mix: 400, 878, 1568 and 2800 µg/plate
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: DMSO has been accepted and approved by authorities and international guidelines. A solubility test was performed based on visual assessment. the test item was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves until the test item had completely dissolved.
Analysis of test item in vehicle for concentration, stability, homogeneity was not performed, however, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Formulations were visually inspected for homogeneity prior to use and all formulations were used within 1.5 hours after adding vehicle to the test item.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9; 2.5 µg/plate in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9; 10 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9; 10 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
in DMSO; 2.5 μg/plate for TA1535 (5% + 10% S9-mix) and TA1537 (5% S9-mix); 5 μg/plate for TA1537 (10% S9-mix); 1 μg/plate for TA98 (5% + 10% S9-mix) and TA100 (5% S9-mix); 2 μg/plate for TA100 (10% S9-mix); 15 μg/plate for WP2uvrA (5% + 10% S9-mix).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

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

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Since in the second mutation assay in tester strain TA98 in the presence of S9-mix, no toxicity and no precipitate on the plates was observed at the highest dose levels tested, an additional mutation experiment was performed. In addition, not enough non-toxic dose levels were present in the tester strain TA1537 in the absence of S9-mix, therefore this tester strain was also tested in the additional experiment.
Rationale for test conditions:
A dose-range finding test was performed and the highest concentration of the test item used in the subsequent mutation assay was 5000 μg/plate or the level at which the test item inhibited bacterial growth.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) 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 Charles River Den Bosch.
b) 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.
c) 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.

INTERPRETATION
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test substance is considered positive if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle 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:
No formal hypothesis testing was done.

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
Vehicle controls validity:
valid
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
Vehicle controls validity:
valid
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
Vehicle controls validity:
valid
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
Vehicle controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item on the plates was not observed at the start of the incubation period in any tester strain. At the end of the incubation period the test item precipitated at 5000 μg/plate in the dose range finding study only.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 164 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA no toxicity was observed up to and including 5000 μg/plate in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- 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.

HISTORICAL CONTROL DATA
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 - 1381 78 - 1058 55 – 1311 55 – 1051 410 – 1995 250 - 1907
Mean 828 218 686 376 1270 883
SD 151 109 320 142 338 340
n 1875 1829 1560 1716 1766 1851

TA100 WP2uvrA
S9-mix - + - +
Range 554 – 1848 408 - 2651 112 – 1951 85 - 1359
Mean 892 1352 1165 388
SD 174 342 488 152
n 1820 1857 1506 1557


- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 5 - 36 3 - 32 3 – 23 3 – 23 8 - 41 9 - 52
Mean 12 12 6 8 16 23
SD 5 4 3 4 5 7
n 1865 1862 1740 1715 1852 1912

TA100 WP2uvrA
S9-mix - + - +
Range 66 - 156 65 - 154 10 – 56 9 - 69
Mean 100 100 25 31
SD 15 16 6 7
n 1853 1877 1571 1583

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 52 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 400 µg/plate and above
TA1537: without S9: 50 µg/plate and above; with 5%S9: 164 µg/plate and above; with 10% S9: 400 µg/plate and above
TA98: without S9: 164 µg/plate and above; with 5% S9: 512 µg/plate and above; with 10% S9: 878 µg/plate and above
TA100: without S9: 100 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 400 µg/plate and above
WP2uvrA: toxicity was observed up to and including 5000 µg/plate

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 guideline and in accordance with GLP principles, X-19924 was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.
Executive summary:

In an AMES test, performed according to OECD 471 guideline and in accordance with GLP principles, X-19924 was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.