Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 - 25 Sep 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction from rat
Test concentrations with justification for top dose:
Toxicity test:
50, 158, 500, 1580 and 5000 µg/plate

Main Assay I:
5000, 2500, 1250, 625 and 313 µg/plate (-S9)
1600, 800, 400, 200, 100 µg/plate (+S9)

In Main Assay II, the test item was assayed at the same dose levels used in Main Assay I.
Vehicle / solvent:
The test item was used as a solution in acetone.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone, DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80 °C in RTC. Overnight subcultures of these stocks were prepared for each day’s work PRELIMINARY TOXICITY TEST A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

MAIN EXPERIMENTS Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures. The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube. The incubate was vortexed and placed at 37 °C for 30 minutes. 2 mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

INCUBATION AND SCORING The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates were scored by counting the number of revertant colonies on each plate.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Results and discussion

Test resultsopen allclose all
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
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
TOXICITY TEST
The test item TMP pelargonate was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration in the absence of metabolic activation and at the two highest concentrations in the presence of metabolic activation. No toxicity was observed with any tester strain at any dose level, both in the absence or presence of S9 metabolism.

ASSAY FOR REVERSE MUTATION
Two experiments were performed. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate (-S9) and 1600, 800, 400, 200, 100 μg/plate (+S9). No toxicity was observed with any tester strain at any dose level both in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations, both in the absence and presence of metabolic activation. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentrations in the presence of S9 metabolism only. No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative