Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 October 2011 - 12 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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
Qualifier:
according to
Guideline:
other: Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment.
Version / remarks:
revised November 20, 2006
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: Off-white powder
- Test item storage: In refrigerator (2-8°C) in the dark

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.
Test concentrations with justification for top dose:
Experiment 1
Without and with S9-mix: 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines.
- The stock solutions were treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were used within 3 hours after preparation.

Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO: 1 μg/plate for TA98 (5 and 10% S9) and for TA100 (5% S9), 2.5 μg/plate for TA1535 (5 and 10% S9) and for TA1537 (5% S9) and for TA100 (10% S9), 5 μg/plate for TA1537 (10% S9), 10 μg/plate for WP2uvrA (5 and 10% S9).
Remarks:
With S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 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 were recorded.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
of 5000 μg/plate
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:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
of 5000 μg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
of 5000 μg/plate
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:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
of 5000 μg/plate
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, but tested up to precipitating concentrations
Remarks:
of 5000 μg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 1000 µg/plate and upwards.

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 245 - 1270 60 – 943 89 - 1086 82 - 677 401 - 1342 225 - 1656
Mean 887 265 314 374 1016 792
SD 112 111 146 104 133 286
n 1123 1150 1011 1039 1136 1157

TA100 WP2uvrA
S9-mix - + - +
Range 348 - 1417 229 - 1752 138 - 1479 122 - 1248
Mean 950 1103 1074 372
SD 117 261 212 137
n 1153 1170 1023 1047

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between June 2008 and June 2011.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 - 25 3 – 31 2 - 19 2 - 16 11 - 49 12 - 59 61 - 195 58 - 179 8 - 45 6 - 46
Mean 10 8 5 5 19 24 106 88 22 23
SD 4 3 2 2 5 7 22 21 7 7
n 1208 1216 1073 1098 1191 1208 1224 1221 1090 1105

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between July 2008 and June 2011.

- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 guideline and GLP principles, ROC-118 was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles. All bacterial strains (TA98, TA100, TA1535, TA1537 and WP2uvrA) showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity of the substance was observed. Precipitation of the test substance was observed at concentrations of 1000 µg/plate and upwards. 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.

Based on the results of this study it is concluded that ROC-118 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.