Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Strudy preparation (information completion, study plan): 09/05/2018 to 28/05/2018. Experimental part of study: 01/06/2018 to 22/06/2018. Examination of results and final report completion: 04/06/2018 to 08/08/2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Liver homogenate
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors (NADP and glucoso-6-phospate).
Test concentrations with justification for top dose:
CYTOTOXICITY EXPERIMENTS: Cytotoxicity test, which serves for finding of optimal ocncentrations for the mutagenity test, was performed in strain TA 100, without metabolic avtivation. Concentrations used for the cytotoxicity experiments were 5000, 2500, 1000, 500 and 10 µg per plate.
THE FIRST MUTAGENICITY EXPERIMENTS: The first mutagenicity experiment was performed with and without metabolic activation with concentration row of concentrations 3000, 1000, 300, 100,
30 and 10 (Salmonella typhimurium TA 98 only) µg per plate.
THE SECOND MUTAGENITY EXPERIMENTS: The second mutagenicity experiments were not performed because positive result was found in the first experiments.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 0.1 mL of DMSO
Details on test system and experimental conditions:
CYTOTOXICITY EXPERIMENTS: Cytotoxicity test, which serves for finding of optimal concentration for the mutagenicity test, was performed in strain TA 100 in duplicate for each dose level (two
plates) using procedure analogous to the ones described in the mutagenicity experiments without metabolic activation (no S9).
THE FIRST MUTAGENICITY EXPERIMENTS: 100 µL of the test item with a required dose level (concentration), 100 µL of 16-18 h culture of tester strain of density 10^8-10^9 CFU /mL, 0.5 mL relevant buffer, and 30 or 100 µL of S9 post mitochondrial fraction (only for the metabolic activation samples) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at
45 ± 3°C. After shaking, the mixture was poured into a minimal glucose agar plate. Three plates were used for each test item dose level.
Petri dishes were incubated for 72 h at 37 ± 1 °C, the number of revertant colonies on the plate was counted manually for all plates except the ones with positive controls, which were counted using an AccuCount 1000.
Fresh suspensions/solutions of the test item were prepared before each experiment. In all experiments, tubes with application forms of the test item as well as tubes with top agar were shaken before any handling.

Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Ri/Rc is reached.
An increase is considered as " biologica lly relevant" :
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion > 10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤ 10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evalua tion of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
conc. 3000 μg per plate
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
prelimitary cytotocixity experiments
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined

Any other information on results incl. tables

THE PRELIMINARY CYTOTOXICITY EXPERIMENTS

The test item solubility in DMSO was higer than the highest dose level of 5000 μg/plate (0.1 mL/plate) recommended by the OECD TG 47. Therefore, the preliminary toxicity experiment included the highest dose level sample, which was then diluted to generate 5 addition doses to cover a dose range from 10 to 5000 μg/plate. The doses were tested for toxicity in strain TA 100 without metabolic activation. Precipitation (bubbles) occurred in molten top agar stating from 2500 μg per plate. Signs of cytotoxicity occured in Petri dishes starting form 2500 μg per plate. Concentration of 3000 μg per plate was chosen as the maximum concentration for mutagenicity testing.

Applicant's summary and conclusion

Conclusions:
The test item, Pentyl nitrite, was non-mutagenic for E.coli WP2uvrA and S. typhimurium TA 98, TA 1537 and TA 100 without and with metabolic activation.
The test item was mutagenic for S. typhimurium TA 1535 detecting of vase-pair substitution mutations without and with metabolic activation.