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Toxicological information

Genetic toxicity: in vitro

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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:
From 19 March 2019 to 15 April 2019
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed

Method

Target gene:
his, trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: S. typhimurium TA1535 hisG46 rfa uvrB, S. typhimurium TA1537 hisC3076 rfa uvrB, S. typhimurium TA98 hisD3052 rfa uvrB pKM101, S. typhimurium TA100 hisG46 rfa uvrB pKM101, E. coli WP2 trpE uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from livers of phenobarbital sodium & 5,6-benzoflavone exposed rats
Test concentrations with justification for top dose:
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:
Strain TA 100: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate

The maximum concentration (5000 μg/plate) is the OECD TG 471 maximum recommended dose level.
Vehicle / solvent:
ethanol (purity > 99%)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD 2-aminoanthracene, 2-AA
Remarks:
Without metabolic activation
TA 1535, TA 100 - sodium azide; TA 1537, TA 98 - 4-nitro-o-phenylene-diamine; WP2 uvrA - methyl methane sulfonate

With Metabolic Activation
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA - 2-aminoanthracene
Details on test system and experimental conditions:
The S9 fraction was from the livers of rats, dosed orally with triple treatments (consecutive days) of 80 mg/kg body weight phenobarbital and B-Naphthoflavone. The S9 fraction was purchased from a commercial source and stored at -80°C.

S9-Mix and Agar:
- An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix (8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 4 mM NADP) in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. The S9 mix substitution buffer contains per litre: 700 mL 100 mM sodium-ortho-phosphate-buffer pH 7.4; 300 mL KCl solution 0.15 M. During the experiment, the S9 mix and S9 mix substitution buffer was stored in an ice bath.

-The following materials were mixed in a test tube and incubated at 37°C for 60 minutes:
50 (experiment II) or 100 (experiment 1) μL Test solution at each dose level (solvent control) or 100 μL Reference mutagen solution (positive control); 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation); 100 μL Bacteria suspension (cf. 3.4.3 Precultures).
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark. This procedure was repeated, in triplicate, on the day of each experiment.

Media:
- Plates with selective agar (without histidine/tryptophan) were used.

Data Recording:
-The colonies were counted using a validated computer system (cf. 3.8, Major computerized systems), which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to precipitation of the test item and reduced background growth the colonies were partly counted manually.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.

An increase of revertant colonies equal or above 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.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiment I at 5000 μg/plate without S9 mix and from 1000 to 5000 μg/plate with S9 mix, and in experiment II at 5000
Vehicle controls validity:
valid
Untreated negative 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 the study substance at any concentration 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. They showed a distinct in-crease in induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

This study was performed to investigate the potential of the study substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.


The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:


Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


Experiment II:


Strain TA 100: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate


The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate with and without S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate without S9 mix and from 1000 to 5000 μg/plate with S9 mix. The undissolved particles had no influence on the data recording.


The plates incubated with the study substance showed reduced background growth in experiment I in strains TA 1537 and TA 100 with and without S9 mix, and in experiment II in all strains with S9 mix and in strains TA 1537 and TA 100 without S9 mix.


Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiment I at 5000 μg/plate without S9 mix and from 1000 to 5000 μg/plate with S9 mix, and in experiment II at 5000 μg/plate with and without S9 mix. In the remaining strains no toxic effects were observed, neither in the presence nor absence of metabolic activation


No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the study substance 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.