Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
(1997)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: granular
Details on test material:
Batch no. RFR 7027-5
Specific details on test material used for the study:
- Stability under test conditions: A stability test in the formulation at 0.3 and 100 mg/mL in ethanol revealed no significant degradation of the test item up to at least 24 hours (Ref. No. XV72LK).

Method

Target gene:
Histidine-deficient mutant  strains of Salmonella typhimurium LT2
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:
S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced rats
Test concentrations with justification for top dose:
Pre-test for cytotoxicity: doses of 3 to 5000 µg/plate were tested (recommended maximum test concentration) in experiment I. Since no relevant toxic effects were observed 5000 μg/plate were chosen as maximal concentration.
The following concentrations were tested in experiment II:
Strain TA 1537: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other:
Remarks:
Na-azide, 4-NOPD, and MMC only used without S9 mix, 2-AA only used with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A pre-test for cytotoxicity/Exp. I was conducted as plate incorporation, the independent repeat/Exp. II as pre-incubation modification (Pre-incubation time 60 minutes at 37°C). Each strain and concentration was tested in triplicate.

DETERMINATION OF CYTOTOXICITY:
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: S. typhimurium TA1535, TA1537, TA98, and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Minor toxic effects were observed in experiment II in strain TA 100 with and without S9 mix at 5000 μg/plate. No other toxic effects occurred in the remaining test groups with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with RFR 6634 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.


TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed reduced background growth in Exp. I in strain TA 1537 with S9 mix from 2500 to 5000 μg/plate and in Exp. II in strains TA 1537 and TA 98 without S9 mix at 5000 μg/plate. In the remaining test groups with and without S9 mix normal background growth was observed on the incubated agar plates.
Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in Exp. II in strain TA 100 with and without S9 mix at 5000 μg/plate. No other toxic effects occurred in the remaining test groups with and without metabolic activation.

Applicant's summary and conclusion

Executive summary:

A bacterial reverse mutation test (Ames), equivalent to OECD TG 471, was conducted for the evaluation of point mutagenic effects. In this assay four histidine-deficient mutant strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA and 98) and the E.coli WP2 uvrA strain were used, with and without a metabolic activating system. The study was conducted as initial plate incorporation with the pre-incubation modification as independent repeat. Doses in the pre-experiment ranged from 3 to 5000 μg/plate.

No precipitation of the test item occurred up to the highest investigated dose.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in experiment II in strain TA 100 with and without S9 mix at 5000 μg/plate. No other toxic effects occurred in the remaining test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.