Fast Pyrolysis Bio-oil

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Fast Pyrolysis Bio-oil was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. In another experiment, in vitro micronucleus assay was carried out in order to investigate Fast Pyrolysis Bio-oil for a possible potential to induce micronuclei in Chinese hamster V79 cells. In these studies, the test substance showed mutagenic activity. Both studies were GLP compliant guideline studies. Also in BIOTOX-project, a guideline in vitro mutagenicity testing was conducted to fast pyrolysis bio-oil (BIOTOX-21). Also in this study, the test substance showed mutagenic activity.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one mutagenicity experiment was performed

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

Target gene:

LT2

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-13 to 2013-05-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IWGT Recommendations

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
50, 150, 500, 1000, 2000 and 5000 µg/mL

Main Experiment:
without metabolic activation:
2, 5, 10, 25, 50, 70, 90 and 110 µg/mL
and with metabolic activation:
25, 50, 90, 110, 130, 150, 170 and 190 µg/mL

Vehicle / solvent:

According to the results of the solubility test DMSO was used as solvent for the test item stock solution. From the highest test item stock solution separate stock solutions were prepared for each of the concentrations by serial dilution in DMSO. From these DMSO stock solutions 1% v/v was added to the cells in RPMI + 5% HS. After adding the stock solutions to the cells in RPMI cell culture medium a light brown discolouration of the cell culture medium was noted. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

1.5 µg/mL

Positive control substance:

ethylmethanesulphonate

Remarks:

300 µg/mL

Positive control substance:

methylmethanesulfonate

Remarks:

10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in dimethylsulfsoxide (DMSO)
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS:one main experiment with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

Evaluation criteria:

The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Additionally a hint for clastogenicity was observed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
positive Additionally a hint for clastogenicity was observed.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Fast Pyrolysis Bio-oil is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells. Additionally a hint for clastogenicity was observed.

Executive summary:

The test item Fast Pyrolysis Bio-oil was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment without metabolic activation 110 µg/mLand with metabolic activation 190 µg/mL were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

DMSO was used as solvent for the test item stock solution. From the highest test item stock solution separate stock solutions were prepared for each of the concentrations by serial dilution in DMSO.

The test item was investigated at the following concentrations:

without metabolic activation:

2, 5, 10, 25, 50, 70, 90 and 110 µg/mL

and with metabolic activation:

25, 50, 90, 110, 130, 150, 170 and 190 µg/mL

No precipitation of the test item was noted in the pre- and main experiment.

Growth inhibition was observed in the main experiment without and with metabolic activation.

In the main experiment without metabolic activation the relative total growth (RTG) was 15.9% for the highest concentration (110 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 190 µg/mL with a RTG of 14.3%.

In the main experiment a biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was exceeded by the induced mutant frequency at several concentrations.

A dose-response relationship was observed.

Additionally, in the main experiment colony sizing showed clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisifes the requirements for Test Guidelines OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: clastogenicity/aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-02 to 2013-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Pre-Experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0, 2500.0 and 5000.0 µg/mL
Main Experiment:
without metabolic activation: 10, 30, 50 and 65 µg/mL
with metabolic activation: 30, 60, 170 and 200 µg/mL

Vehicle / solvent:

- Justification for choice of solvent/vehicle:
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium. Therefore the test item was dissolved in dimethylsulfoxide (DMSO) (brown solution, light protected) and diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples. The pH-value of the test item solution diluted in cell culture medium was within the physiological range. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

clastogenic control without metabolic activation [1200 µg/mL]

Positive controls:

yes

Positive control substance:

other: colcemide

Remarks:

aneugenic control without metabolic activation [1.0 µg/mL]

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

clastogenic control with metabolic activation [2.5 µg/mL ]

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Main Experiment with and without metabolic activation)

FIXATION INTERVAL:
24 hours (Main Experiment)

STAIN (for cytogenetic assays): acridin orange

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2000 cells per concentration (1000 cells per culture)

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI)

Evaluation criteria:

There are several criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells containing micronuclei
- a biologically relevant increase in the number of cells containing micronuclei for at least one of the dose groups, which is higher than the laboratory negative control range.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.

Statistics:

Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: positive

In conclusion, it can be stated that during the study described and under the experimental conditions reported, Fast Pyrolysis Bio-oil did induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, Fast Pyrolysis Bio-oil is considered to be mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate Fast Pyrolysis Bio-oil for a possible potential to induce micronuclei in Chinese hamster V79 cells in vitro a micronucleus assay was carried out. The test item was dissolved in dimethylsulfoxide (DMSO) (brown solution, light protected) and diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples. The selection of the concentrations was based on data from the pre-experiment. In the main experiment without metabolic activation 200 µg/mL and with metabolic activation 250 µg/mL test item were selected as the highest concentrations. The following concentrations were evaluated for micronucleus frequencies: Main Experiment: without metabolic activation: 10, 30, 50 and 65 mg/mL with metabolic activation: 30, 60, 170 and 200 mg/mL No precipitate of the test item was noted in all dose groups evaluated in the pre- and in the main experiment without and with metabolic activation with the unaided eye. However, precipitation of the test item was seen in the main experiment without metabolic activation at a concentration of 150 µg/mL and higher and in the main experiment with metabolic activation at a concentration of 200 µg/mL and higher by microscopical evaluation. According the OECD Guideline 487 the maximum of cytotoxicity should not exceed the limit of 55% ± 5%. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. According to laboratory experience a culture showing reduced cell viability (less than 70% rel. CBPI) of the negative control displays cytotoxicity. Due to this the acceptable limit of cytotoxicity is ≥ 30%.This corresponds to ≤ 70% of rel. CBPI. In the main experiment without metabolic activation no decrease of the relative CBPI below 70 % was noted up to a concentration of 30 µg/mL. At a concentration of 50 µg/mL a relative CBPI of 63% and at a concentration of 65 µg/mL a relative CBPI of 46% was noted. In the main experiment with metabolic activation no decrease of the relative CBPI below 70 % was noted up to a concentration of 150 µg/mL. At a concentration of 170 µg/mL a relative CBPI of 67% and at a concentration of 200 µg/mL a relative CBPI of 54% was noted. In the main experiment without and with metabolic activation a biologically relevant increase of the micronucleus frequency was noted after treatment with the test item. The nonparametricc² Test was performed to verify the results of the main experiment. A statistically significant enhancement ( p < 0.05) of cells with micronuclei was noted at concentrations of 50 μg/mL and 65 μg/mL in the main experimentwithout metabolic activation. A statistically significant enhancement (p < 0.05) of cells with micronuclei was also noted at concentrations of 30 μg/mL,170 μg/mL and 200 μg/mL of the test item in the main experimentwith metabolic activation. These values were significantly increased compared to the corresponding solvent control and the micronucleated cell frequencies wereabove the historical control data range of the negative control. Based on these data, the number of micronucleated cells found in some groups treated with the test item showed a biologically relevant increase compared to the corresponding solvent control. Ethylmethanesulfonate (EMS, 1200 μg/mL) and Cyclophosphamide (CPA, 2.5 μg/mL) were used as clastogenic controls. Colcemide (1.0 μg/mL) was used as aneugenic control. All induced distinct and biologically relevant increases of micronucleus frequency. This demonstrates the validity of the assay.This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487 for In Vitro Mammalian Cell Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo testing for mutagenicity (Test MAS In vivo: Bone marrow micronucleus test by oral route) of fast pyrolysis bio-oil in BIOTOX project gave negative results. Based on in vivo mutagenicity testing Fast Pyrolysis Bio-oil is not considered to be mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01.01.2003-30.06.2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Guideline study, final study report not available. Results published in BIOTOX project (BIOTOX. 2005. An assessment of bio-oil toxicity for safe handling and transportation. Final Technical Report. Part I: Publishable Final Report. Report drafted by Blin, J. Project N°: S07.16365. Project co-ordinator: Centre de Coopération Internationale de Recherche Agronomique pour le Développement (Cirad). Partners: Aston University, Bundesforschungsanstalt für Forst- und Holzwirtschaft (BFH)).

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Commission Directive No. 2000/32/EC, B12, 8 June 2000

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Swiss, Ico: OF1 (IOPS Caw).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Upon their arrival at CIT, the animals were housed in an animal room, with the following environmental conditions: temperature: 22 ± 2°C . relative humidity: 30 to 70% . light/dark cycle: 12 h/12 h (07:00 - 19:00) . ventilation: at least 12 cycles/hour of filtered non-recycled fresh air. The temperature and relative humidity are under continuous control and recording. The housing conditions (temperature, relative humidity and ventilation) and corresponding instrumentation and equipment are verified and calibrated at regular intervals. The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France).

Route of administration:

oral: unspecified

Vehicle:

Corn oil

Details on exposure:

For the vehicle and the test item:
. Route: oral, since it is a possible route of exposure in Man,
. Frequency: two treatments separated by 24 hours
. Volume: ≤ 20 mL/kg.
For the positive control (CPA):
. Route: oral
. Frequency: one treatment,
. Volume: 10 mL/kg,

Duration of treatment / exposure:

48 h

Frequency of treatment:

Twice, separated by 24 h

Post exposure period:

24 h

Remarks:

Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide 50 mg/kg

Tissues and cell types examined:

Blood, bone marrow

Details of tissue and slide preparation:

The femurs were removed and the bone marrow was flushed out with fetal calf serum. After centrifugation, the supernatant were removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation (“blind” scoring).

Evaluation criteria:

For each animal, the number of micronucleated polychromatic erythrocytes (MPE) were counted, the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). The analysis of the slides was performed at CIT or at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK).
For a result to be considered positive, there must be: a statistically significant increase in the frequency of MPE when compared to the vehicle control group. Reference to historical data, or other considerations of biological relevance may be taken into account in the evaluation of data obtained.

Statistics:

MPE in each treated group were compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the χ2 value (Lovell et al, 1989) (d). If there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969) (e). The student "t" test was used for the PE/NE ratio comparison. Probability values of p ≤ 0.05 was considered as significant.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No bone marrow toxicity (the ratio of polychromatic and normochromatic erythrocytes did not change.

Vehicle controls validity:

valid

Negative controls validity:

other: only vehicle controls

Positive controls validity:

valid

Additional information on results:

No significant increase in the frequency of micronucleated cells was observed in animals treated with the bio-oil sample. Analyses on blood samples would be necessary to confirm inactivity of the bio-oil sample.

No significant increase in the frequency of micronucleated cells was observed in animals treated with the bio-oil sample. Analyses on blood samples could not be carried out due to highly complex nature of the test item and the lack of suitable kinetic indicators. However, highest dose was as high as 2000 mg/kg/day (given twice), and in the acute toxicity study and 7-day repeated toxicity study clear systemic effects were observed (NOAEL 150 mg/kg/day). Therefore, it is likely that after the high exposure levels of the present study the components of test item are present in circulation and therefore also in the bone marrow.

Conclusions:

Interpretation of results: negative
No significant increase in the frequency of micronucleated cells was observed in animals treated with the bio-oil sample.

Executive summary:

In the MAS test the potential of bio-oil to induce damage to the chromosomes or mitotic apparatus in bone marrow cells of mice (after 2 oral administrations) was assessed in vivo. No significant increase in the frequency of micronucleated cells was observed in animals treated with the bio-oil sample at the dose levels up to 2000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vivo testing for mutagenicity (Test MAS In vivo: Bone marrow micronucleus test by oral route) of fast pyrolysis bio-oil in BIOTOX project gave negative results.No significant increase in the frequency of micronucleated cells was observed in animals treated with the bio-oil sample. Analyses on blood samples could not be carried out due to highly complex nature of the test item and the lack of suitable kinetic indicators. However, highest dose was as high as 2000 mg/kg/day (given twice), and in the acute toxicity study and 7-day repeated toxicity study clear systemic effects were observed (NOAEL 150 mg/kg/day). Therefore it is likely that after the high exposure levels of the present study the components of test item are present in circulation and therefore also in the bone marrow.

Justification for selection of genetic toxicity endpoint
in vivo guideline study

Justification for classification or non-classification

In vivo testing for mutagenicity (Test MAS In vivo: Bone marrow micronucleus test by oral route) of fast pyrolysis bio-oil in BIOTOX project gave negative results. Consequently, classification as a germ cell mutagen is not justified. Moreover, the amount of components classified as category 2 mutagens (formaldehyde) is under 0,5 %.