Fast Pyrolysis Bio-oil

Genetic toxicity: in vitro

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:

1 March 2005 - 18 March 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one mutagenicity experiment was performed

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, B.13., 8 June 2000

Deviations:

yes

Remarks:

only one mutagenicity experiment was performed

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fast Pyrolysis Bio-oil (BIOTOX-21)

IUPAC Name:

Fast Pyrolysis Bio-oil (BIOTOX-21)

Test material form:

liquid: viscous

Details on test material:

Test item: BIOTOX-21
Appearance: brown liquid
purity: 100 %
Date of receipt: 21 February 2005
Storage conditions: at + 4 °C and protected from light

Method

Target gene:

LT2

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Five dose levels of bio-oil
without S9 mix: 250, 500, 625, 750, 1000 and 1250 µg/plate, for all tester strains
with S9 mix: 250, 500, 625, 750, 1000 and 1250 µg/plate, for the TA 1535, TA 1537, TA 98 and TA 100 strains;625, 750, 1000, 1250, 2500 and 3750 µg/plate, for the TA 102 strain

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

The bacterial strains were stored in cryoprotective medium in liquid nitrogen. The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.
The test item was tested in a preliminary test and one mutagenicity experiment. The preliminary test and the mutagenicity experiment were performed according to the direct plate incorporation method.

Evaluation criteria:

a 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the (TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance were taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

PRELIMINARY TOXICITY TEST

The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL. Consequently, with maximum treatment volume of 50 µL/plate, the dose levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate. No precipitate was observed in the plates at any dose level. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and / or a thinning of the bacterial lawn.

Without S9 mix:

A moderate to marked toxicity was observed at dose levels ≥2500 µL/plate in the TA 98, TA 100 and TA 102 strains.

With S9 mix:

A moderate to marked toxicity was observed at dose levels ≥2500 µL/plate in the TA 98 and TA 100 strains and a marked toxicity was observed at 5000 µL/plate in the TA 102 strain.

MUTAGENICITY EXPERIMENT:

Without S9 mix:

The selected treatment levels were: 250, 500, 625, 750, 1000 and 1250 µg/plate, for all tester strains. No toxicity was observed at any dose level. Dose-related increases in the number of revertants were noted in the TA 98 and TA 102 strains.

With S9 mix:

The selected treatment levels were: 250, 500, 625, 750, 1000 and 1250 µg/plate, for the TA 1535, TA 1537, TA 98 and TA 100 strains;625, 750, 1000, 1250, 2500 and 3750 µg/plate, for the TA 102 strain.

A moderate toxicity was observed at 3750 µg/plate in the TA 102 strain. Dose-related increases in the number of revertants were noted in the TA 98, TA 100 and TA 102 strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive

Under experimental conditions, the test item showed mutagenic activity:
without S9 mix, in Salmonella typhimurium TA 98 and TA 102 strains
with S9 mix, in Salmonella typhimurium TA 98, TA 100 and TA 102 strains

Executive summary:

The potential of test item Fast Pyrolysis Bio-oil (BIOTOX-21) to induce reverse mutation in Salmonella typhimurium was evaluated in a guideline study Bacterial reverse mutation test. The test was performed in the absence and presence of a rat liver metabolizing system (S9 mix). The validity criteria of test were fulfilled. Under experimental conditions, the test substance showed mutagenic activity.