Docosane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No key or supporting data is available for docosane. However, key data is available from a structurally related substance, C18-C50 branched, cyclic and linear hydrocarbons – Distillates (CAS# 848301-69-9) and is presented below:

In vitro:
- Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains (TA 98, 100, 1535, 1537) and Escherichia coli WP2uvrA (OECD 471).
- Cytogenicity in mammalian cells: negative with and without activation in human lymphocytes (OECD Draft Guideline 487).
- Cytogenicity in mammalian cells: negative with and without activation in human lymphocytes (OECD 473).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of the study was performed between 22 February 2006 and 31 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Annex V of 2000/32/EC

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98 and TA100. E. coli: WPuvrA-

Metabolic activation:

with and without

Metabolic activation system:

Naphthoflavone/phenobarbitone induced, rat-liver S9.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 0, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solvent: tetrahydrofuran

Untreated negative controls:

yes

Remarks:

2-Aminoanthracene (2-AA); Benzo(a)pyrene (BP)

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrafuran

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG); 9-Aminoacridine (9AA); 4-Nitroquinoline-1-oxide (4NQO)

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 1500 µg/plate

Species / strain:

other: Salmonella typhimurium strains TA100 and Escherichia coli strains WP2uvrA-

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium strains TA100 and Escherichia coli strains WP2uvrA-

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strains WP2uvrA-

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strains WP2uvrA-

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
Solvent control plates gave counts of revertant colonies within the normal range. All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges. No statistically significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the substance, either with or without metabolic activation. The substance was found to be non-mutagenic under the conditions of this test.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative with metabolic activation Not classified with regards to potential germ cell mutagenicity according to Regulation (EC) No) 1272/2008
negative without metabolic activation Not classified with regards to potential germ cell mutagenicity according to Regulation (EC) No) 1272/2008

non-mutagenic (with and without S9)

Executive summary:

The substance 'Distillates (Fischer-Tropsch), heavy, C18-50, branched, cyclic and linear' has been tested in a bacterial mutagenicity study according to OECD 471 and under GLP using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA. The test material was dissolved in tetrahydrofuran at concentrations up to 5000 µg/plate. Appropriate solvent and positive controls were included and gave expected results. No toxicity to bacterial cells was observed. No significant increase in the number of revertants was observed at any concentration with and without metabolic activation in any of the strains tested. The results were confirmed in a repeat experiment; both experiments used the direct plate incorporation method.

The test material was considered to be non-mutagenic under the conditions of the tests both with and without S9.

Reference 2

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

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: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

Not applicable.

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 .

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)

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.

Key result

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'.

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

Conclusions:

Interpretation of results: negative

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.

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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 10 May 2006 and 19 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

other: Draft OECD Guideline 487 modified in line with OECD Guideline 473

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Naphthoflavone/phenobarbitone induced, rat-liver S9.

Test concentrations with justification for top dose:

Experiment 1:
Without S9- 0, 160, 320, 640 µg/plate
With S9- 0, 160, 320, 640 µg/plate

Experiment 2:
Without S9- 625, 1250, 2500 µg/plate
With S9- 625, 1250, 2500 µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Remarks:

Mytomycin and demecolcine (-S9): Cyclophosphamide (+S9)

Positive control substance:

other: Mytomycin and demecolcine (-S9): Cyclophosphamide (+S9)

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

>625µg/mL

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitating concentration>=156.25µg/mL with S9

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

2500µg/mL

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitating concentrations: >640µg/ mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Preliminary

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 9.75 to 2500µg/mL. The maximum dose was based on the maximum practical dose level. An cloudy precipitate of the test material was observed in the parallel blood-free cultures at the the end of the exposure at and above 78.1µg/mL in both exposure groups without S9 and at and above 156.25µg/mL in the presence of S9. The precipitate was generally observed to form a greasy/ oily layer at and above 625µg/mL in the whole blood cultures, though there was some variation between exposure groups, and it was considered that overall the maximum exposure to the cells was occurring at about this dose level. Microscopic assessment of the slides prepared from the cultures showed that binucleate cells were present at up to 2500µg/mL in all three of the exposure groups. The CBPI data are presented in Table 1.

The test material induced no evidence of toxicity in any of the exposure groups.

Micronucleus Test- Experiment 1

The dose levels of the controls and the test material selected for binucleate analysis were 0, 160, 320 and 640 µg/mL in Groups 4(16) hour without S9 and 4(16) hour with S9.

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of the test material investigated, 640µg/L in the absence and presence of S9. Precipitate observations taken in the Preliminary Toxicity Test were considered to be representative for the study.

The CBPI data are given in Table 2. They confirm the qualitative observations in that no marked dose-related inhibition of CBPI was observed in either the absence or presence of S9. The maximum dose level selected for analysis of binucleate cells was the maximum dose level investigated (640µg/mL).

The micronucleus frequency data are given in Table 4 and 5. The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive cntrol materials induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test material itself was operating as expected.

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

Experiment 2

The dose levels of the controls and the test material selected for binucleate analysis were 0, 160, 320 and 640 µg/mL in Groups 20- hour without S9 and 4(16)- hour with S9.

The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the maximum test material dose level investigated, 640µg/mL, in both the absence and presence of S9. Precipitate was observed at and above 80µg/mL, in the absence of S9, and at and above 160µg/mL in the presence of S9.

The CBPI data are given in Table 3. They confirm the qualitative obsrevations in that there was no dose-related inhibition of CBPI observed in eithe the absence or presence of S9. The maximum dose level selected for binucleate cell analysis was the maximum dose level investiagtes (640µg/mL).

The micronucleus data are given in Table 6 and 7. The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test matertial did not induce any statistically significant increases in the frequency of cells with micronuclei, in either the absence or presence of metabolic acivation system.

Conclusions:

Interpretation of results:
negative without metabolic activation Not classified with regards to potential germ cell mutagenicity according to Regulation (EC) No) 1272/2008
negative with metabolic activation Not classified with regards to potential germ cell mutagenicity according to Regulation (EC) No) 1272/2008

non-clastogenic and non-aneugenic to human lymphocytes in vitro

Executive summary:

An in vitro micronucleus study has been conducted using the substance ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ following OECD draft guideline 487 and conducted under GLP conditions. No increase in the incidence of micronuclei was observed in duplicate cultures of human lymphocytes at any concentration in either the initial experiment (4 hour exposure, 16 hour expression, with and without metabolic activation) or the repeat experiment (20 hour exposure without metabolic activation; 4 hour exposure, 16 hour expression, with metabolic activation). No test material induced toxicity was observed. The test material was dissolved in acetone, and the maximum concentration tested was 2500 µg/plate; higher concentrations could not be tested due to difficulties in formulating the test material in the vehicle. The vehicle controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes, and appropriate positive controls were concluded and induced significant increases in the number of cells with micronuclei.

It was concluded that the test material is non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No key or supporting data is available for docosane. However, key data is available from a structurally related substance, C18-C50 branched, cyclic and linear hydrocarbons – Distillates (CAS# 848301-69-9) and is presented below:

In vivo:

- Cytogenicity: negative in Mammalian Bone Marrow Chromosome Aberration Test (OECD 474/EU B.11)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 26 January 2011 and 14 March 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

At the start of the test the rats weighed 184 to 231 g and were approximately seven to twelve weeks old. After a minimum acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
The animals were housed in groups of up to five in solid-floor polypropylene cages with woodflake bedding (Datesand Ltd, Cheshire, UK.). Free access to mains drinking water and food, Harlan Teklad 2014 Rodent Pelleted Diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK, was allowed throughout the study. The animals were also provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) which were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

Supplier's identification: Arachis oil
Serial number (laboratory): V-4855
Date received: 14 June 2010
Description: Straw-coloured slightly viscous liquid
Expiry date: 31 January 2012
Storage conditions: Room temperature

Details on exposure:

Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours. In addition, two further groups of rats were included in the study; one group of seven rats was dosed via the oral route with the vehicle alone (arachis oil) and a second group of five rats was dosed orally with Cyclophosphamide.
Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test. The vehicle control group and positive control group were killed approximately 24 hours following treatment.
All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later. The experimental design is summarised as follows:

Treatment Group Dose Level(mg/kg) Concentration (mg/ml) Dose Volume (ml/kg) Kill Time (Hours After Dosing) Animal Numbers
1. Vehicle Control (Arachis oil) 0 0 10 24 1 – 7
2. Positive Control (Cyclophosphamide) 25 2.5 10 24 8 – 12
3. Test item 2000 200 10 48 13 – 19
4. Test item 2000 200 10 24 20 – 26
5. Test item 1000 100 10 24 27 – 33
6. Test item 500 50 10 24 34 – 40

Frequency of treatment:

Groups of rats were dosed once only via the oral route

Post exposure period:

All animals were observed for signs of overt toxicity and death approximately one hour after dosing and then once daily as applicable and immediately prior to termination.
Treatment with Mitotic Inhibitor
Animals were injected via the intraperitoneal route with a solution of Colchicine at 4 mg/kg 2 to 4 hours prior to bone marrow harvest. At thescheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue.

Remarks:

Doses / Concentrations:
Groups, each of seven rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg.
Basis:
other: The maximum recommended dose of 2000 mg/kg was used as the maximum dose, substance was formulated in Arachis oil at 50, 100 and 200 mg/ml and dosed at 10 ml/Kg to achieve actual dose levels.

No. of animals per sex per dose:

Groups, each of seven male rats were dosed once only via the oral route with the test item at 2000, 1000, and 500 mg/kg. One group of male rats from each dose level was killed by cervical dislocation approximately 24 hours following treatment and a second group dosed at 2000 mg/kg was killed at approximately 48 hours.

Control animals:

yes, concurrent vehicle

Positive control(s):

Five rats were dosed orally with Cyclophosphamide at a dose of 25 mg/kg. Cyclophosphamide is a positive control item known to produce chromosome aberrations under the conditions of the test.
The positive control item was supplied by Acros Organics, as follows:
Supplier's identification : Cyclophosphamide
Supplier’s lot number : A0277203
Harlan serial number : R-4723
Date received: 04 December 2009
Expiry date : 04 December 2011
Storage conditions: Approximately 4ºC, in the dark

Tissues and cell types examined:

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in rat bone marrow cells. In addition any increases in polyploidy may indicate the induction of numerical aberrations

Details of tissue and slide preparation:

At the scheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue. The bone marrow was aspirated into 5 ml of warm Hanks buffered salt solution (HBSS) supplemented with demecolcine (Colcemid 0.1 µg/ml) and incubated at approximately 37°C for 30 minutes before being spun down in a centrifuge. The supernatant was removed and the cell pellet re-suspended in 0.075 M potassium chloride (KCl) at 37°C for approximately 15 minutes including centrifugation. The cells were re centrifuged and all but 1 ml of the supernatant removed. After re-suspension of the cell pellet, the cells were fixed by the addition of freshly prepared fixative (methanol:glacial acetic acid, 3:1). The fixative was changed several times and the cells stored at approximately 4ºC for at least 4 hours.
Slide Preparation and Staining
After storage the cell suspensions were centrifuged and the fixative removed to leave a sufficient amount to give a milky suspension on re-suspension of the cell pellet. A few drops of each cell suspension were dropped onto clean, wet slides and air-dried. When completely dry the slides were stained in 5% Giemsa for 10 minutes and rinsed in tap water and distilled water. When the slides were dry a cover slip was applied using a mounting medium.

Evaluation criteria:

Slide Evaluation
The stained slides were coded and examined ‘blind’ using light microscopy at x100 and x1000 magnifications. 100 metaphase cells of adequate quality were scored, if possible, from the slides prepared from each animal for both numerical and structural chromosome aberrations. Except where there were approximately 30 to 50% of cells with aberrations, then slide evaluation was terminated at 50 cells. A mitotic index (MI) value was also obtained for each animal by recording the number of metaphase cells that were associated with 1000 cells.
If the cell had 40 to 44 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS Guidelines for Mutagenicity Testing. The details of the classification of chromosome aberrations and the evaluation criteria applied to test data are given in Appendix 1. A Senior Cytogeneticist checked aberrations recorded by the slide scorers.

Statistics:

Statistical Analysis
Comparisons were made between the vehicle control group and each treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations. Analysis of mitotic index data was performed using a Students T-Test following a √(x+1) transformation.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A summary of the results of the chromosome aberration test is given in the attached Table 1. Individual and group mean data are presented in the attached Tables 2 to 7.
No marked decreases in the mitotic index mean value were observed with any of the test item groups or the positive control group when compared to the vehicle control group.
All of the vehicle control animals gave values of chromosome aberrations within the expected range (attached Table 2).
The positive control group animals showed highly significant increases in the frequency of aberrations (attached Table 3) indicating that the test method itself was operating as expected. It should be noted that due to the toxic response seen with cyclophosphamide in the bone marrow the quality and morphology of the metaphases was less than perfect. Therefore it was necessary to score more than 100 metaphase cells for three of the five animals to demonstrate the sensitivity of the rats following exposure with cyclophosphamide.
There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group.
The test item did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups.

Due to the nature and format of the tables, please see the attached results tables.

Conclusions:

Interpretation of results: negative
The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non clastogenic to rat bone marrow cells in vivo.

Executive summary:

Introduction. 

The study was performed to assess the potential of the test item ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ to produce damage to chromosomes or the mitotic apparatus when administered to rats. The method used is designed to be compatible with that described in the revised OECD Guidelines for Testing of Chemicals No. 475 “Mammalian Bone Marrow Chromosome Aberration Test”, Method B11 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 andUS, EPA, TSCA and FIFRA guidelines.

 

Methods. 

A range-finding test was not performed as the test item had been previously investigated at a dose of 5000 mg/kg with no ill effects. Therefore, the maximum recommended dose of 2000 mg/kg was used as the maximum dose and at the request of the Sponsor only male animals were investigated via the oral route.

The chromosome aberration test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, processed and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.

Further group of rats for the 24-hour time point were given a single oral dose of Arachis oil (seven rats) or dosed orally with Cyclophosphamide (five rats) to serve as vehicle and positive controls respectively.

Results. 

There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any dose level.

No marked decreases in the mitotic index mean value were observed in any of the test item dose groups when compared to the vehicle control group.

There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group. 

The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in any of the exposure groups.

The positive control item produced a marked increase in the frequency of chromosome aberrations.

Conclusion. 

The test item did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non-clastogenic to rat bone marrow cells in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No key or supporting data is available for docosane. However, key data is available from a structurally related substance, C18-C50 branched, cyclic and linear hydrocarbons – Distillates (CAS# 848301-69-9) and is presented below:

C18-C50 branched, cyclic and linear hydrocarbons – Distillates (CAS# 848301-69 -9) has been tested in a bacterial mutagenicity study according to OECD 471 and under GLP using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA. The test material was dissolved in tetrahydrofuran at

concentrations up to 5000 µg/plate. Appropriate solvent and positive controls were included and gave expected results. No toxicity to bacterial cells was observed. No significant increase in the number of revertants was observed at any concentration with and without metabolic activation in any of the strains tested. The results were confirmed in a repeat experiment; both experiments used the direct plate incorporation method.

An in vitro micronucleus study has been conducted using GTL Base Oil Distillates following OECD draft guideline 487 and conducted under GLP conditions. No increase in the incidence of micronuclei was observed in duplicate cultures of human lymphocytes at any concentration in either the initial experiment (4 hour exposure, 16 hour expression, with and without metabolic activation) or the repeat experiment (20 hour exposure without metabolic activation; 4 hour exposure, 16 hour expression, with metabolic activation). No test material induced toxicity was observed. The test material was dissolved in acetone, and the maximum concentration tested was 2500 µg/plate; higher concentrations could not be tested due to difficulties in formulating the test material in the vehicle. The vehicle controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes, and appropriate positive controls were concluded and induced significant increases in the number of cells with micronuclei. It was concluded that the test material is non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Further evidence of the lack of effects on chromosomes in vitro was obtained when the substance was tested according to OECD 473 and under GLP. No statistically significant increase in the frequency of cells with chromosome aberrations was observed in either the initial or the repeat experiment when tested with and without metabolic activation up to a dose level that was limited by the onset of precipitate. Appropriate solvent and positive controls were included and gave expected results.

Moreover, in vivo data is available from an in vivo chromosome aberration study on C18 -C50 branched, cyclic and linear hydrocarbons - Distillates (CAS# 848301 -69 -9) conducted according to OECD 475 and under GLP:

The test was conducted using the oral route in groups of seven rats at the maximum recommended dose (MRD) 2000 mg/kg for the 24-hour and 48-hour time points, with 1000 and 500 mg/kg as the lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, processed and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.

 

There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any dose level. No marked decreases in the mitotic index mean value were observed in any of the test item dose groups when compared to the vehicle control group. There was no evidence of a statistically significant increase in the incidence of cells with chromosome aberrations excluding gaps in animals dosed with the test item, when the dose groups were compared to the vehicle control group. 

 

The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in any of the exposure groups and it did not induce any significant or dose-related increases in the frequency of chromosome aberrations. The test item was considered to be non-clastogenic to rat bone marrow cells in vivo.

Justification for classification or non-classification

Based on the available read across in vitro and in vivo data, Docosane is not genotoxic and does not require classification according to Regulation 1272/2008/EC.