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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study.
Justification for type of information:
Lead data provides genetic toxicity in vitro information

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
UK Department of Health, 15 October 2007
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
NEXBTL renewable diesel
NEXBTL renewable diesel
Details on test material:
- Name of test material (as cited in study report): NExBTL Renewable diesel
- Description: clear colourless liquid
- Date received: 18 December 2007


Species / strain
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:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: ethylmethanesulphonate (150 ug/ml), cyclophosphamide (2 ug/ml)
Details on test system and experimental conditions:
- Exponentially growing cells

- 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

- 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)

- 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).
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).

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables


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 µg/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.



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


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

Applicant's summary and conclusion

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.