Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2009-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 471, EPA OPPTS 870.5100, EU Method B 13/14 and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): UCAR n-pentyl propionate
- Physical state: liquid
- Analytical purity: 99.973%
- Purity test date: June 25, 2009
- Lot/batch No.: WL0901FTCl
- Storage condition of test material: Ambient (+I8 to +36 °C)

Method

Target gene:
The purpose of this study was to evaluate the mutagenic potential of UCAR n-Pentyl Propionate and/or its metabolites by utilizing a pre-incubation method to measure its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA pKM101 in the presence and absence of S9 activation.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation
Test concentrations with justification for top dose:
Initial Toxicity Mutation assay - 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Confirmatory Mutation assay - 100, 266, 707, 1880 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: recommended vehicle by various regulatory agencies
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, 2-Aminoanthracene, 9-Aminoacridine, 4-Nitroquinoline-1-oxide and sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

The petri-dishes were labeled to indicate the study number, strain number, treatment group, trial number, plate number and activation. For the initial toxicity mutation test two replicates were used while for the confirmatory mutation test three replicates were used. Mutation assay was performed following the pre-incubation procedure. The following test constituents were transferred into sterile test tubes and were kept in an incubator shaker for approximately 20 minutes at 37 °C and 110 rpm. After this period, soft agar containing histidine-biotin 1 tryptophan (2 mL) was added to each of the tubes.

A. For the test in the presence of metabolic activation -
a) 100 µL test concentration/solvent/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL S-9 mix

B. For the test in the absence of metabolic activation -
a) 100 µL test concentration/solvent/appropriate positive control
b) 100 µL bacterial culture
c) 500 µL of PBS

The tube contents were mixed and overlaid onto VB agar plates. After the soft agar had set, the plates were incubated at 37 ± 1°C for 67 hours. After incubation, the revertant colonies in each plate were counted manually and the plates were examined for bacterial background lawn. All these procedures were carried out under yellow light.

Active ingredient analyses was performed on samples from all the dose levels along with the DMSO control.

The bacterial suspension of each tester strain was diluted up to 10-6 dilution in PBS. One hundred microlitres from the 10-6 dilution of each tester strain was
plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1°C for 67 hours. After incubation, the number of colonies in each plate were manually counted and expressed as the number of colony forming units per mL of the bacterial suspension.
Evaluation criteria:
The conditions necessary for determining a positive result were that there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.
For strains TA98, TA1535, and TA1537, data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean vehicle control value.
For strains TAl00 and WP2 uvrA (pKM101), data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean vehicle control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Statistics:
Standard statistical methods were employed

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Initial Toxicity – Mutation Assay -
The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean numbers of revertant colonies was comparable to or lower than that of the respective solvent control plates at all the tested concentrations, both in the presence and absence of metabolic activation. The test article did not precipitate the basal agar plates in any of the tested concentrations, either in the presence or in the absence of metabolic activation. The intensity of the bacterial background lawn for TA 98, TA 1535 and TA 1537 strains of Salmonella was comparable to that of the respective solvent control plates at all the tested concentrations except at 5000 µg/plate, wherein moderate thinning of the bacterial background lawn was observed both in the presence and absence of metabolic activation. At 5000 µg/plate, for Salmonella strain TA 100 and for the E. coli strain, there was a slight reduction in the bacterial background lawn in the presence of metabolic activation and a moderate reduction in the absence of metabolic activation.
Positive control chemicals tested simultaneously produced a significant increase in the mean numbers of revertant colonies for all the strains when compared to the respective solvent control plates. The intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective solvent control plates.

Confirmatory Mutation Assay -
The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains. For all the tester strains, the mean numbers of revertant colonies was comparable to or lower than that of the respective solvent control plates at all the tested concentrations, both in the presence and absence of metabolic activation. The test article did not precipitate the basal agar plates in any of the tested concentrations, either in the presence or in the absence of metabolic activation. The intensity of the bacterial background lawn for TA 98, TA 1535 and TA 1537 strains of Salmonella was comparable to that of the respective solvent control plates at all the tested concentrations except at 5000 µg/plate, wherein moderate thinning of the bacterial background lawn was observed both in the presence and absence of metabolic activation. For Salmonella strain TA 100 and for the E. coli strain, there was a slight reduction in the bacterial background lawn both in the presence and absence of metabolic activation. Positive control chemicals tested simultaneously produced a significant increase in the mean numbers of revertant colonies for all the strains when compared to the respective solvent control plates. The intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective solvent control plates.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The S-9 homogenate was found to be active and sterile and the protein content was found to be 25 mg/ml.Salmonella typhimuriumstrains TA 98, TA 100, TA 1535 and TA 1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia colistrain WP2 uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA 98, TA 100 and WP2 uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all theSalmonella typhimuriumstrains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coJistrain and the uvrB mutation in theSalmonella typhimuriumstrains was demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility's historical control data. The test article was stable in DMSO for 24 hours at 15 and 50000 µg/ml and was homogeneous in DMSO at both the doses. The most concentrated test article dilution, the sham (PBS) and S9 mixes were found to be sterile. Viable counts of all the tester strains were within the required range of 1-2 x109CFU/ml for both the trials of the mutation assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the study, UCAR n-pentyl propionate was not considered mutagenic in this bacterial reverse mutation assay at the highest tested concentration of 5000 μg/plate.
Executive summary:

UCAR n-pentyl propionate was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA 98, TA 100, TA 1535 and TA 1537 strains of Salmonella typhimurium and WP2 uvrA (pKM 101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test article in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using preincubation procedure.

UCAR n-pentyl propionate was found to be soluble in dimethyl sulphoxide (DMSO) at the required concentration of 50000 μg/mL.

In the initial toxicity-mutation test, UCAR n-pentyl propionate was exposed in duplicate at the concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate along with the solvent and appropriate positive controls. The test article did not cause any precipitation on the basal agar plates up to the highest tested concentration of 5000 μg/plate, but exhibited toxicity to the bacterial tester strains in terms of reduction in mean revertant colonies as well as thinning of bacterial background lawn at the highest tested concentration of 5000 μg/plate compared to the solvent control, in the presence and absence of metabolic activation.

Based on these initial findings, in the confirmatory mutation test, the test article was tested in triplicate at the concentrations of 100, 266, 707, 1880 and 5000 μg/plate along with the solvent and appropriate positive controls, in the presence and absence of metabolic activation. The mean and standard deviation of revertant colonies were calculated for each test concentration and the controls for all the tester strains.

The results showed that the mean numbers of revertant colonies neither doubled for strains TA 100 and WP2 uvrA (pKM101) nor tripled for strains TA 98, TA 1535 and TA 1537 when compared to the respective solvent control plates, either in the presence or in the absence of the metabolic activation at any of the tested concentrations. In this study, a significant increase in the mean numbers of revertant colonies in the positive controls was seen, demonstrating assay sensitivity.

The study indicated that the test article, UCAR n-pentyl propionate, was not mutagenic in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay up to the highest tested concentration of 5000 μg/plate.