Fast Pyrolysis Bio-oil

Genetic toxicity: in vivo

Administrative data

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)).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Test guideline 2

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

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:

BIOTOX 21: Biomass: Spruce, produced with Fluidised bed at 500 °C

Test animals

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).

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.