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:
2014.09.15~2014.12.30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.015, 0.03, 0.06, 0.125, 0.25, 0.5, 1 µg/plate
Vehicle / solvent:
distilled water, DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Remarks:
All strains with and without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(-5-nitro-2-furyl)-acrylamide
Remarks:
TA100, TA98, WP2 uvrA Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(-5-nitro-2-furyl)-acrylamide
Remarks:
TA98 Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene(2-AA)
Remarks:
All strains with S9 mix
Details on test system and experimental conditions:
3.8.1 Selection of the test method
This test was carried out using the pre-incubation method under the presence and absence of S9 mix.
3.8.2 Pre-incubation Method
(1) For each treatment, 0.1 mL of the test substance solution, negative (solvent) control or positive control solution was added into a sterilized test tube.
(2) For assays in the absence of S9 mix, 0.5 mL of 0.1 mol/L sodium phosphate buffer (pH 7.4) was mixed and 0.1 mL of a bacterial suspension was added to this mixture.
(3) For assays in the presence of S9 mix, 0.5 mL of S9 mix was mixed and 0.1 mL of a bacterial suspension was added to this mixture.
(4) The mixture was incubated with gentle shaking (Shaking frequency: 120 times/minute) for 20 minutes at 37°C (pre-incubation).
(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and then poured onto a minimal glucose agar plate.
(6) After the overlaid agar had solidified, the plates were incubated for (48 ± 2) hours at 37°C.
3.8.3 Observation
Precipitation : After incubation for (48 ±2) hours, presence of precipitation on the agar plates was examined with the unaided eye.
Microbial toxicity : After incubation for (48 ± 2) hours, microbial toxicity was examined with a stereoscopic microscope.
3.8.4 Colony count
The colonies in each plate were counted with the unaided eye.
3.8.5 Number of plate
concentration range-finding test: 3 plate/dose
Main test: 3 plate/dose
3.8.6 Expression of data
The mean of the measured number of revertant colonies was calculated for the negative
(solvent) control, the positive control and the test substance treatments. The mean was
expressed by rounding to the first decimal place.
3.8.7 Sterility test
The sterility test was also performed, using one plate for each of the highest dose of test substance solution and the S9 mix used in the test.
(1) In the concentration range-finding test and main test, 0.1 mL of the highest dose of the test substance solution or 0.5 mL S9 mix was mixed with 2 mL of the molten top agar.
(2) The mixture was poured onto a minimal glucose agar plate.
(3) After the overlaid agar had solidified, the plates were incubated for (48 ± 2) hours at 37°C, and bacterial contamination was checked with the unaided eye.
3.8.8 Acceptable criteria of the test
The test was accepted as valid if all the following criteria were satisfied.
(1) The negative (solvent) control values (mean value) and the positive control values (mean value) were within the proper ranges calculated based on the historical data at the testing facility.
(2) The positive control values (mean value) show positive responses in the respective test strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (solvent) control value.
(3) There are 4 or more doses giving no microbial toxicity and at least 5 analyzable doses.
(4) The highest dose in the main test is conducted up to the doses at which microbial toxicity was observed or 5000 μg/plate.
(5) The result of the sterility test indicates that there is no microbial contamination.
(6) There are no plates, which became invalid for measurement due to contamination or other unexpected situations.
Evaluation criteria:
Test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependent increase in the number of the revertant colonies (mean value) to a level equal to or greater than 2-fold of the negative (solvent) control value (mean value) in each test strains either in the presence of absence of S9 mix and also when the dose-dependent increase was reproducible. Otherwise, the mutagenicity of the test substance was considered to be negative. No statistical analysis was performed with the test results in the present study.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Main test
The results of the main tests were summarized in tables 4 ~ 5 and appendices 4 ~ 5. In the two main tests, growth inhibitions were observed at the dose of 1 μg/plate or more for TA98, WP2uvrA, and at the dose of 0.5 μg/plate or more for TA100 TA1535 and TA1537 in the absence and presence of S9 mix.
The number of revertant colonies from the test substance-treated group did not show an increase surface when compared to the negative control group. Precipitation was not observed on the agar plates at the any dose in the presence and absence of S9 mix.

Sterility test
Microbial contamination was not observed in the highest dose of test substance solution and S9 mix in the sterility test group in either the concentration range-finding tests or the main tests.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that Red 620 was not mutagenic (negative) under the conditions employed in the present study.