Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Version 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: room temperature, darkness (Optimal temperature at 20°C)

Method

Target gene:
Histidine gene for Salmonella and tryptophan gene for E.coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 37°C for 48 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 48 hours at about 37 ºC, the number of colonies per plate was counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation). The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The most concentrated test article treatment formulation, vehicle, S-9 mix and buffer solutions will be checked for sterility by plating the same volume used for treatment onto nutrient agar plates.
- Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility.

Evaluation criteria:
For valid data, the test article will be considered to be mutagenic in this assay if:
1. A concentration related increase in revertant numbers is 2.0-fold (in strains TA98, TA100 or TA102) or 3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response is reproducible under the same treatment conditions

The test article will be regarded positive in this assay if both of the above criteria are met.
The test article will be regarded negative in this assay if neither of the above criteria are met.
A Dunnett’s test will be used to confirm results.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.
Statistics:
A Dunnett’s test will be used to confirm the results.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
not specified
Remarks:
On going
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
on going
Vehicle controls validity:
not specified
Remarks:
on going
Untreated negative controls validity:
not specified
Remarks:
on going
True negative controls validity:
not applicable
Positive controls validity:
not specified
Remarks:
on going
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
not specified
Remarks:
on going
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
on going
Vehicle controls validity:
not specified
Remarks:
on going
Untreated negative controls validity:
not specified
Remarks:
on going
True negative controls validity:
not applicable
Positive controls validity:
not specified
Remarks:
on going
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
not specified
Remarks:
on going
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
on going
Vehicle controls validity:
not specified
Remarks:
on going
Untreated negative controls validity:
not specified
Remarks:
on going
True negative controls validity:
not applicable
Positive controls validity:
not specified
Remarks:
on going
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
not specified
Remarks:
on-going
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
on-going
Vehicle controls validity:
not specified
Remarks:
on-going
Untreated negative controls validity:
not specified
Remarks:
on-going
True negative controls validity:
not applicable
Positive controls validity:
not specified
Remarks:
on-going
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
not specified
Remarks:
on going
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
on going
Vehicle controls validity:
not specified
Remarks:
on going
Untreated negative controls validity:
not specified
Remarks:
on going
True negative controls validity:
not applicable
Positive controls validity:
not specified
Remarks:
on going

Applicant's summary and conclusion

Conclusions:
The results of this test are not available (on-going).
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and TA 102 were exposed the test material in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Negative and positive control groups were also included in mutagenicity tests.

The results of this test are not available (on-going).