[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 17 May 2010 and 26 July 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: is acceptable to the Japanese New Chemical Substance Law (METI)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-220-0

EC Name:

-

Cas Number:

848301-69-9

IUPAC Name:

C18-C50 branched, cyclic and linear hydrocarbons – Distillates

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test
The dose range of test material used was 9.77 to 2500 µg/ml.

Experiment 1:

Group Final concentration of Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear (µg/ml)
4(20)-hour without S9 0*, 2.5, 5, 10, 20*, 40*, 80*, MMC 0.4*
4(20)-hour with S9 0*, 2.5, 5, 10, 20*, 40*, 80*, CP 5*

Experiment 2:

Group Final concentration of Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear (µg/ml)
24-hour without S9 0*, 2.5, 5, 10, 20*, 40*, 80*, MMC 0.2*
4(20)-hour with S9 0*, 2.5, 5, 10, 20*, 40*, 80*, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

Tetrahydrofuran (THF) was selected as the solvent because the test material was soluble in it at the required concentrations .

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran (THF)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 Migrated to IUCLID6: (CP)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran (THF)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material dissolved in THF
- Precipitation:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/ml. The maximum dose was the maximum practical dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of exposure, at and above 39.06 µg/ml, in the 4-hour exposure group in the presence of S9 and in the 24 hour exposure group in the absence of S9. In the 4-hour exposure group in the absence of S9 precipitate was observed at and above 78.13 µg/ml. The precipitate became greasy/oily at and above 625 µg/ml in the 4-hour exposure groups and at and above 312.5 in the 24-hour continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 µg/ml in all three of the exposure groups. The mitotic index data are presented in the attached Appendix 1 (5) and (6). The test material induced no clear evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the main experiments was based on the onset of precipitate in all exposure groups and was limited to a maximum dose level of 80 µg/ml.

EXPERIMENT 1:
The dose levels of the controls and the test material are given in the table below:

Group Final concentration of Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear (µg/ml)
4(20)-hour without S9 0*, 2.5, 5, 10, 20*, 40*, 80*, MMC 0.4*
4(20)-hour with S9 0*, 2.5, 5, 10, 20*, 40*, 80*, CP 5*

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum test material dose level of 80 µg/ml in both the absence and presence of metabolic activation (S9).
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 1, Appendix 2. These data show that an approximate 50% growth inhibition was not achieved in either the absence or presence of S9.
A greasy/oily precipitate of the test material was observed at the end of the treatment period at 80 µg/ml in the absence of S9 only.
The maximum dose level selected for metaphase analysis was based on the lowest precipitating dose level as seen in the preliminary toxicity test in both the absence and presence of S9, and was the maximum dose level investigated of 80 µg/ml.
The chromosome aberration data are given in the attached Form 1, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9).
The polyploid cell frequency data are given in the attached Form 1, Appendix 2. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.


EXPERIMENT 2:
The dose levels of the controls and the test material are given in the following table:

Group Final concentration of Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear (µg/ml)
24-hour without S9 0*, 2.5, 5, 10, 20*, 40*, 80*, MMC 0.2*
4(20)-hour with S9 0*, 2.5, 5, 10, 20*, 40*, 80*, CP 5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to the maximum test material dose level of 80 µg/ml in both the absence and presence of S9.
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 2, Appendix 2. These data show that an approximate 50% growth inhibition was not achieved in either the absence or presence of S9.
A cloudy precipitate of the test material was observed at the end of the treatment period at and above 40 µg/ml in the absence of S9 which formed a greasy/oily precipitate at 80 µg/ml. No precipitate was observed at any dose level in the presence of S9.
The maximum dose level selected for metaphase analysis was the same as in Experiment 1, and was the maximum dose level tested (80 µg/ml).
The chromosome aberration data are given in the attached Form 2, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.
The polyploid cell frequency data are given in the attached Form 2, Appendix 2. The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Due to the nature and format of the results, please refer to the attached tables and dose response curves

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/ml. The maximum dose was the maximum practical dose level.  A precipitate of the test material was observed in the parallel blood-free cultures at the end of exposure, at and above 39.06 µg/ml, in the 4-hour exposure group in the presence of S9 and in the 24 -hour exposure group in the absence of S9. In the 4-hour exposure group in the absence of S9 precipitate was observed at and above 78.13 µg/ml. The precipitate became greasy/oily at and above 625 µg/ml in the 4-hour exposure groups and at and above 312.5 in the 24-hour continuous exposure group.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 µg/ml in all three of the exposure groups. The mitotic index data are presented in the attached Appendix 1 (5) and (6). The test material induced no clear evidence of toxicity in any of the exposure groups.

The selection of the maximum dose level for the main experiments was based on the onset of precipitate in all exposure groups and was limited to a maximum dose level of 80 µg/ml.

Chromosome Aberration Test – Experiment 1

The dose levels of the controls and the test material are given in the table below:

Group	Final concentration of Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) (µg/ml)
4(20)-hour without S9	0*, 2.5, 5, 10, 20*, 40*, 80*, MMC0.4*
4(20)-hour with S9	0*, 2.5, 5, 10, 20*, 40*, 80*, CP5*

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum test material dose level of 80 µg/ml in both the absence and presence of metabolic activation (S9). The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 1, Appendix 2.  These data show that an approximate 50% growth inhibition was not achieved in either the absence or presence of S9. A greasy/oily precipitate of the test material was observed at the end of the treatment period at 80 µg/ml in the absence of S9 only. The maximum dose level selected for metaphase analysis was based on the lowest precipitating dose level as seen in the preliminary toxicity test in both the absence and presence of S9, and was the maximum dose level investigated of 80 µg/ml.

The chromosome aberration data are given in the attached Form 1, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9).

The polyploid cell frequency data are given in the attached Form 1, Appendix 2. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test material are given in the following table:

Group	Final concentration of Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear (µg/ml)
24-hour without S9	0*, 2.5, 5, 10, 20*, 40*, 80*, MMC0.2*
4(20)-hour with S9	0*, 2.5, 5, 10, 20*, 40*, 80*, CP5*

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to the maximum test material dose level of 80 µg/ml in both the absence and presence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 2, Appendix 2. These data show that an approximate 50% growth inhibition was not achieved in either the absence or presence of S9.

A cloudy precipitate of the test material was observed at the end of the treatment period at and above 40 µg/ml in the absence of S9 which formed a greasy/oily precipitate at 80 µg/ml. No precipitate was observed at any dose level in the presence of S9.

The maximum dose level selected for metaphase analysis was the same as in Experiment 1, and was the maximum dose level tested (80 µg/ml). The chromosome aberration data are given in the attached Form 2, Appendix 2.  All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in the attached Form 2, Appendix 2. The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

CONCLUSION

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

*Dose levels selected for metaphase analysis

MMC= Mitomycin C

CP= Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Interpretation of results: negative

Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of test material(µg/ml)
4(20)-hour without S9	2.5, 5, 10, 20, 40, 80
4(20)-hour with S9 (2%)	2.5, 5, 10, 20, 40, 80
24-hour without S9	2.5, 5, 10, 20, 40, 80
4(20)-hour with S9 (1%)	2.5, 5, 10, 20, 40, 80

Results. 

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was generally considered to be non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included a dose level that was limited by the onset of precipitate.

Conclusion. 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.