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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK Department of Health, 13 February 2003
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): NExBTL Biodiesel
- Description: Clear colourless liquid
- Date received: 04 July 2006

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: obtained from University of California at Berkley on 4 August 1995
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: obtained from University of California at Berkley on 4 August 1995
Metabolic activation:
with and without
Metabolic activation system:
Adult male Sprague Dawley rat liver S9 fraction (induced with three consecutive daily doses of 80 mg/kg bw/d phenobarbitone, 100 mg/kg bw/d B-naphthoflavone)
Test concentrations with justification for top dose:
Following test concentrations used (based on preliminary toxicity test with TA100): 0, 50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: based on preliminary solubility experiments
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
other: strain-specific positive control substances used in the absence and presence of S9: see below for full details
Details on test system and experimental conditions:
METHOD OF APPLICATION
- In agar (plate incorporation), with triplicate plates prepared per exposure concentration for each experiment

DURATION
- Exposure duration: 48 hr at 37 degrees C

NUMBER OF EXPERIMENTS:
- 2 independent repeats

DETERMINATION OF CYTOTOXICITY
- Method: TA100 exposed to 0.15-5000 ug/plate (10 concentrations) by plate incorporation, +/- S9, for 48 hr at 37 degrees C. An oily precipitate was observed at > 1500 μg/plate, but this was not toxic to the bacteria.
Evaluation criteria:
Dose-related increase in revertant frequency over the dose range tested, and/or a dose-related increase at one or more concentrations in at least one bacterial strain with or without metabolic activation.

Results and discussion

Test results
Species / strain:
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 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:
CYTOTOXICITY: The test substance was practically non-toxic to TA100.

STUDY RESULTS: No increase in the frequency of revertant colonies was recorded for any of the tester strains, both in the absence and in the presence of S9 fraction.

POSITIVE CONTROLS: a satisfactory response was obtained with all of the positive control substances.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Not mutagenic in 5 standard Salmonella typhimurium tester strains (TA100, TA1535, TA1537, TA102, or TA98) in the absence or presence PB/B-NF-induced rat S9 fraction.
Executive summary:

Mutagenic potential assessed in a GLP-compliant guideline bacterial mutation assay (reverse mutation assay, method B13/14 of directive 2004/73/EC) using Salmonella typhimurium tester strains TA100, TA1535, TA1537, TA102 and TA98 exposed via plate incorporation. Based on results obtained from an initial toxicity screen, test concentrations of 50-5000 ug/plate were used in the absence and in the presence of phenobarbitone/B-naphthaflavone induced rat S9 fraction, and the study run using 2 independent repeats. An oily precipitate was observed at 1500 μg/plate and above, but this was not toxic to the bacteria. There was no significant increase in the frequency of revertant colonies in any strain both with and without metabolic activation. The results indicate that NExBTL renewable diesel is not mutagenic in this bacterial assay (Ames test) when tested up to the maximum concentration required in current test guidelines.