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Genetic toxicity in vitro

Description of key information

The genotoxicity of NExBTL renewable diesel has been assessed in guideline mutation studies performed in vitro using 5 strains of Salmonella typhimurium (bacterial reverse mutation assay; method B13/14 of directive 2004/73/EC), human lymphocytes (chromosomal aberration test; method B10 of directive 2004/73/EC) and L5178Y TK cells (mammalian cell mutation assay; method B17 of directive 2004/73/EC). Testing was conducted at the maximum concentration required by each guideline with the inclusion of appropriate positive control substances and S9 fraction. The studies were GLP-compliant and the information is reliable and fully adequate for evaluation. No evidence of genotoxicity was apparent in any test, and it is concluded that NExBTL renewable diesel is not mutagenic or clastogenic in vitro.

Link to relevant study records

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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
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
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)
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.
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'.
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.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health, 15 October 2007
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone (80/100 mg/kg bw/kg bw/d; 3 consectutive days) induced S9 fraction from male SD rats (approx. 250 g bw)
Test concentrations with justification for top dose:
Experiment 1 and Experiment 2: 0, 40, 80, 160, 320, 480 or 640 ug/ml
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate (150 ug/ml), cyclophosphamide (2 ug/ml)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Exponentially growing cells

DURATION:
- Exposure duration: experiment 1, 4 hr, with and without S9; experiment 2, 24 hr in the absence of S9, 4 hr in the presence of S9
- Exposure temperature: 37 degrees C
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 d

SELECTION AGENT (mutation assays):
- 5-trifluorothymidine

NUMBER OF REPLICATES:
- Incubations performed in duplicate, with and without S9 activation, at 6 dose levels (40-640 ug/ml); the experiment was run twice (i.e two independent repeats)

DETERMINATION OF CYTOTOXICITY:
- Method: relative total growth:
Evaluation criteria:
A response was considered positive if it gave a statistically significant increase in induced mutant frequency over the concurrent vehicle mutant frequency value and if the induced mutation frequency exceeded a Global Evaluation Factor of 126 x 10^-6 (Moore et al. (2006) Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: International Workshop on Genotoxicity Testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environ. Mol. Mutagen. 47(1) pp 1–5).
Statistics:
Data were analysed using a dedicated computer program (Robinson, W D et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140).
Species / strain:
mouse lymphoma L5178Y cells
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

PRELIMINARY TOXICITY TEST:

In all three of the exposure groups there was no significant reduction in the Relative Suspension Growth of cells treated with the test material up to 5000 ug/ml when compared to the concurrent vehicle controls. A cloudy precipitate of the test material was observed at and above 78.13 µg/ml, and a greasy / oily precipitate was formed at and above 625 µg/ml in the 4 hr exposure groups, and at and above 312.5 µg/ml in the 24 hr exposure group. Based on the RSG values for all three of the exposure groups, maximum exposure of the test material to the cells appeared to occur overall at approximately 312.5 to 1250 µg/ml. Therefore, the onset of the greasy/oily precipitate combined with the RSG values resulted in the maximum dose level in the mutagenicity tests being set at 640 µg/ml.

 

MUTAGENICITY TEST:

The maximum dose level used was limited by the onset of a greasy/oily precipitate effectively reducing the exposure of the test material to the cells. Precipitate of test material was observed at and above 40 µg/ml at the end of the exposure period. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

 

Experiment 1:

-S9: mutation frequency range 76.01 to 95.19, linear trend non-significant

+S9: mutation frequency range 53.68 to 97.05, linear trend non-significant

 

Experiment 2:

-S9: mutation frequency range 103.34 to 175.18, linear trend non-significant

+S9: mutation frequency range 65.65 to 86.27, linear trend non-significant

POSITIVE CONTROLS

Experiment 1:

   %RSG  RTG  MF
 EMS, 400 ug/ml -S9  80  0.05  644.2
 CP, 2 ug/ml, +S9  62  0.23  618.05

Experiment 2:

   %RSG  RTG  MF
 EMS, 150 ug/ml -S9  144  1.19  859.93
 CP, 2 ug/ml, +S9  74  0.48  456.57
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Not mutagenic at the TK+/- locus in L5178Y mouse lymphoma cells
Executive summary:

Mutagenic potential was assessed in a GLP compliant guideline study (method B17 of directive 2004/73/EC) using L5178y TK+/- cells. Based on results obtained from an initial cytotoxicity screen, test concentrations of 40-640 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 cloudy precipitate was observed at and above 78.13 ug/ml, and a greasy/oily precipitate formed at and above 625 ug/ml, in the preliminary toxicity test hence the maximum dose level set for the mutation tests was 640 ug/ml. No toxicity was seen up to and including exposure of 5000 ug/ml. No increase in mutation frequency was recorded at any concentration in either experiment. The vehicle (solvent) controls had acceptable mutant frequency values (within normal range) and the positive control substances (-S9 ethylmethylsulphonate; +S9 cyclophosphamide) gave acceptable responses in both experiments. The results indicate that NExBTL renewable diesel is not mutagenic in L5178Y TK+/- cells in vitro.

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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health, 13 February 2003
Type of assay:
bacterial reverse mutation assay
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
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.
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'.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No evidence of genotoxicity in 3 test systems in vitro; no classification required under Directive 67/548/EEC or Regulation EC 1272/2008.