Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 15th to April 7th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 postmitocondrial fraction derived from rat liver, induced by Aroclor 1254 (Concentration of S9 used in the test:10 %)
Test concentrations with justification for top dose:
5, 1.58, 0.5, 0.16, 0.16 and 0.05 mg/plate in main test with and without S9.
In the main test the 5 different bacteria strains were exposed to these 5 concentrations of test substance prepared with a √10 dilution factor. The concentrations were selected according to the preliminary cytotoxicity test performed only on strain TA100 exposed to 5 concentrations. Since no cytotoxicity was observed at 5 mg/plate, the highest concentration allowed in the assay, this solution was used as the first concentration to test in Ames test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO was used as solvent.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 4-Nitro-1,2-phenilendiammine and Nitrofurantoine
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoantracene
Remarks:
with S9
Details on test system and experimental conditions:
SCREENING OF THE STRAIN BEFORE STARTING WITH TEST: the amino-acid requirement for growth (histidine or tryptophane) is always controlled prior to frozen stock culture preparation as well as the resistance to Ampicillin, the sensitivity to crystal violet and the sensitivity to UV light.

CONTAMINATION OF TEST SUBSTANCE: in order to detect microbial contaminations, 100 µl of the test substance solution 50 mg/ml concentrated, prepared in DMSO, were poured in TSA plate, in duplicate, so that the final concentration of the test substance was 5 mg/plate. It was verified that 50 mg/ml solution prepared in DMSO resulted completely soluble. On the other hand, solution prepared in H2O produced precipitates and for this reason it was not used in the test. Plates were incubated for 5 days at 37 °C ± 1 °C. After the incubation, absence of microbial growth was visually verified. No colonies were detected.

PRELIMINARY CITOTOXICITY TEST: Salmonella typhimurium TA100 (one of the 5 strains that are employed in AMES TEST) was exposed to 5 concentrations of the test substance, in the presence and in the absence of an exogenous metabolic activation system (S9 mix).The survival of treated cultures was determined by verifying the presence of a normal bacterial background. Absent or abnormal background was indicative of a cytotoxic response.

AMES TEST: PLATE INCORPORATION METHOD: suspensions of 5 different bacteria strains were exposed to 5 concentrations of the test substance in the presence and in the absence of an exogenous metabolic activation system (S9 mix). The suspensions were then immediately mixed with an overlay agar and plated onto minimal medium. After two days of incubation at 37 °C, revertant colonies were compared to the number of spontaneous revertant colonies on solvent control plates. In parallel the phenotypic characteristics proper of each strain were confirmed repeating the controls of amino-acid requirement, resistance to Ampicillin, the sensitivity to crystal violet and the sensitivity to UV light.

METHOD OF APPLICATION: plate incorporation method.

DURATION: 48 hours at T= 37 °C ± 1 °C.

NUMBER OF REPLICATIONS: 3 for each concentration for each strain with and without S9.

ACCEPTANCE CRITERIA:
- Acceptability criteria for positive controls: in order to be considered mutagenic, positive controls have to show an increase factor ≥ 2 for TA98, TA100 and WP2 strains, and ≥ 3 for TA1535 and TA1537.
- Acceptance criteria for negative controls: negative controls should show a number of revertant colonies between 15 and 40 for TA98, TA1537 and TA1535 and between 80 and 180 for TA100 and WP2.
- Negative and positive controls should be within the acceptable historical range of the laboratory.
Evaluation criteria:
For test strains TA98, TA 100 andWP2 the test substance is considered positive and it is able to induce point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and /or Escherichia coli, when, in at least one of the strains TA98, TA100, WP2 and in at least one condition (+S9/-S9) it will induce an Induction Factor ≥ 2. The increase should also be concentration-related. The dose-response correlation will be verified with Dunnet test (alpha = 0.05 1-sided).

For test stains TA1535, TA1537 the test substance is considered positive and it is able to induce point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli, when in at least one of the strains TA1535, TA1537, in at least one condition (S9/-S9) it produces an induction Factor ≥ 3. The increase should also be concentration-related. The dose-response correlation will be verified with Dunnet test (alpha = 0.05 1-sided).
Statistics:
Mean value and standard deviation (SD) of revertant colonies were calculated for the test solution, the negative and positive controls.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 100, TA 98, TA1535, TA 1537 and E.Coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Preliminary citotoxicity tested only on S. typhimurium TA 100 with and without S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF AMES TEST:
Alter the incubation period, the number of revertant colonies per plate were counted for each dilution of the test substance and for the negative and positive controls. The presence of a bacterial background was also recorded: normal back ground was observed in all plates. Mean value and standard deviation (SD) of revertant colonies were calculated for the test solution, the negative and positive controls. For the test substance and positive controls, the Induction Factor (IF) was calculated as follows: Mean test solution / Mean Respective Negative Control. In all strains the induction factor (IF) did not exceed 2 (TA98, TA100, WP2) or 3 (TA1535, TA1537), hence the substance is not considered mutagenic under the test conditions applied. Notably, it was not necessary to verify the dose-concentration correlation, as no IF increase ≥ 2 or 3 was observed.

RESULT OF PRELIMINARY CITOTOXICITY TEST: No citotoxicity based on background evaluation was in strain TA 100 with and without S9 at any concentration tested of test substance (5, 1.58, 0.5, 0.16 and 0.05 mg/plate). Based on the result of preliminary citotoxicity test the concentration used in the AMES test were selected.

ACCEPTANCE CRITERIA:
- Acceptability criteria for positive controls: fullilled: all positive controls showed an increase factor ≥ 2 for TA98, TA100 and WP2 strains, and ≥ 3 for TA1535 and TA1537.
- Acceptance criteria for negative controls: fullilled: all negative controls showed a number of revertant colonies between 15 and 40 for TA98, TA1537 and TA1535 and between 80 and 180 for TA100 and for WP2.
- Negative and positive controls were within the acceptable historical range of the laboratory.

Applicant's summary and conclusion

Conclusions:
The test item is not a mutagenic agent in a bacterial reverse mutation test with and without metabolic activation.
Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium (TA98, TA100, TA1535, TA 1537) and Escherichia coli WP2 uvrA pKM101 with and without metabolic activation S9 according to OECD TG 471.

In all strains the induction factor (IF) did not exceed 2 (TA98, TA100, ECWP2) or 3 (TA1535, TA1537), hence the substance is not considered mutagenic under the test conditions applied. Notably, it was not necessary to verify the dose-concentration correlation, as no IF increase ≥ 2 or 3 was observed.