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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health, 21 November 2005
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
NEXBTL renewable diesel
IUPAC Name:
NEXBTL renewable diesel
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 / strain
Species / strain / cell type:
lymphocytes: human
Additional strain / cell type characteristics:
not applicable
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 cytotoxicity test ): 0, 312.5, 65, 1250 and 2500 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: based on earlier solubility tests (reported in endpoint study record Genetic Toxicity in vitro.001)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin c (-S9); cyclophosphamide (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In Minimal Essential Medium with duplicate cultures prepared per exposure concentration for each of two independent experiments

NUMBER OF EXPERIMENTS:
- 2 independent repeats

DURATION:
- Preincubation period: 48 hr (to establish primary culture)
- Exposure duration: 4 hr, hower in experiment 2 in incubations in the absence of S9 fraction exposure was continuous for 24 hr
- Expression time (cells in growth medium): experiment 1 (+/- S9) 20 hr; experiment 2 (+S9 only) 20 hr

SPINDLE INHIBITOR (cytogenetic assays):
- Colcemid 0.1 ug/ml (2 hr before harvest)

STAIN (for cytogenetic assays):
- 5% Gurs Giemsa

MITOTIC INDEX:
- 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded; expressed as the mitotic index and as a percentage of the control value.

NUMBER OF CELLS EVALUATED FOR CHROMOSOME DAMAGE:
_ The first 100 consecutive well-spread metaphases from each culture were counted. Slides were coded and scored blind.

DETERMINATION OF CYTOTOXICITY:
- Method: cells were exposed to 9 concentrations of test substance in the range 19.53 - 5000 ug/ml for 4 hr with and without metabolic activation followed by a 20 hr expression period; in one trial without S9 fraction cells received a continuous 24 hr exposure. Mitotic index data were used to estimate cytotoxicity.

OTHER EXAMINATIONS:
- Precipitate observations were performed over the concentration range 19.53 - 5000 ug/ml. An oily precipitate was observed at 1250 ug/ml and above but this was not cytotoxic.

POSITIVE CONTROLS:
- absence of S9 fraction: mitomycin c in Minimal Essential Medium (0.4 ug/ml and 0.2 ug/ml for experiments 1 and 2, respectively)
- presence of S9 fraction: cyclophosphamide in DMSO (4 and 5 ug/ml for experiments 1 and 2, respectively)
Evaluation criteria:
Breaks, gaps and rearrangements were noted according to:
- the International System for Chromosome Nomenclature (1985), ed Harnden, DG et al., Karger, Switzerland
- Savage, JRK (1976) Annotation: classification and relationships of induced chromosomal structural changes. J Med Genet 13, 103-122
- Scott, D et al. (1990) Metaphase chromosome aberration assays in vitro. In: Basic mutagenicity tests - UKEMS recommended procedures, Kirkland DJ, ed. Cambridge University Press
Statistics:
Frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared with concurrent control values using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes:
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 examined
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
- The test substance demonstrated no evidence of cytotoxicity in any of the exposure groups.

STUDY RESULTS:
- No increase in the frequency of cells with aberrations or the incidence of polyploidy was recorded in either experiment in the absence or presence of S9 fraction, up to the maximum dose tested (2500 ug/ml).

POSITIVE CONTROLS:
- A satisfactory response was obtained with the positive control substances in both experiments.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Not clastogenic in human lymphocytes in the absence or presence PB/B-NF-induced rat S9 fraction.
Executive summary:

Clastogenic potential was assessed in a GLP-compliant guideline study (method B10 of directive 2004/73/EC). Based on results obtained from an initial cytotoxicity screen, test concentrations of 312.5-2500 ug/ml 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 1250 ug/plate and above, but this was not cytotoxic. No increase in the frequency of cells with aberrations or the incidence of polyploidy was recorded in two independent experiments in the absence or presence of S9 fraction, up to the maximum dose tested (2500 ug/ml). A satisfactory response was obtained with the positive control substances (-S9 mitomycin c; +S9 cyclophosphamide) in both experiments. The results indicate that NExBTL renewable diesel is not clastogenic to human lymphocytes in vitro.