Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January 2018 - 24 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: Black DB
Appearance: black powder

Method

Target gene:
his/trp
In addition to histidine (his) or tryptophan (trp) mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.
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 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver
Test concentrations with justification for top dose:
initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate
repeated confirmatory mutation test (TA98, TA1535 and TA1537): 50; 16; 5; 1.6 and 0.5 μg/plate.
Justification of doses: Selection of the concentration range in the initial mutation test was done on the basis of the solubility test and concentration range finding tests (informatory toxicity tests). The chosen, already investigated concentration levels were not modified for the confirmatory mutation test. The examined concentration range for the complementary pre-incubation test was chosen based on the confirmatory mutation test results.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: based on solubility test
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes, 37 °C (preincubation method)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants
Evaluation criteria:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was only observed using the preincubation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was only observed using the preincubation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was only observed using the preincubation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 μg/plate in the absence and presence (±S9 mix) of exogenous metabolic activation following the plate incorporation and pre-incubation procedures
-The test item did not show inhibitory, cytotoxic effects in the initial mutation test. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system. Unequivocal inhibitory effect of the test item was observed in the confirmatory mutation test and in the additional complementary pre-incubation test in the investigated Salmonella typhimuriumstrains in the absence of exogenous metabolic activation (-S9 mix). The inhibitory effect was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 16 μg/plate (noticed in S. typhimurium TA1537, in absence of exogenous metabolic activation) was considered as the lowest concentration showing cytotoxicity.
The revertant colony numbers of solvent control dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges

RANGE-FINDING/SCREENING STUDIES: Selection of the concentration range in the initial mutation test was done on the basis of the solubility test and concentration range finding tests (informatory toxicity tests).

Applicant's summary and conclusion

Conclusions:
The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats (the additional complementary pre-incubation test was performed withSalmonella typhimuriumTA98, TA1535 and TA1537, in the absence of S9, only). The study included a preliminary solubility test, preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), a confirmatory mutation test (pre-incubation test) and an additional complementary pre-incubation test. Based on the results of the solubility test and the concentration range finding tests the test item was suspended/dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions a correction of the concentrations for the purity (96.59 %) of test item was made in the experiments. Based on the cytotoxicity and solubility results of the preliminary concentration range finding tests (informatory toxicity tests) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate. The results of the preliminary concentration range finding tests allowed the applying of the recommended maximum test concentration of 5000 μg/plate. Because of the strong inhibitory effect of the test item noticed in the confirmatory mutation test an experimental repeat (completion) with a modified concentration range was considered necessary withS. typhimurium TA98, TA1535 and TA1537 strains in the absence of exogenous metabolic activation (-S9 mix). The examined concentration levels were: 50; 16; 5; 1.6 and 0.5 μg/plate.

When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 μg/plate in the absence and presence (±S9 mix) of exogenous metabolic activation following the plate incorporation and pre-incubation procedures. The test item did not show inhibitory, cytotoxic effects in the initial mutation test. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system. However, unequivocal inhibitory effect of the test item was observed in the confirmatory mutation test and in the additional complementary pre-incubation test in the investigatedSalmonella typhimuriumstrains in the absence of exogenous metabolic activation (-S9 mix). The inhibitory effect was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 16 μg/plate (noticed in S. typhimurium TA1537, in absence of exogenous metabolic activation) was considered as the lowest concentration showing cytotoxicity.

The revertant colony numbers of solvent control dimethyl sulfoxide (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in the revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test itemhas no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.