[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- 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).
- In vitro mammalian cell micronucleus test: negative with and without activation in human lymphocytes (OECD Draft Guideline 487).
- In vitro mammalian chromosome aberration test (on related substances 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Distillates (Fischer-Tropsch), C8-C26, branched and linear' and ' Hydrocarbons, C15-C19, n-alkanes, isoalkanes, < 2% aromatics (EC 940-730-5): negative with and without activation in human lymphocytes (OECD 473).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

justification for analogue approach: see IUCLID section 13 "Read-across justification"

Reason / purpose for cross-reference:

read-across source

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:

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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 no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

August 2005, Certificate dated November 2005

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

other: None

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg / plate
Range-finding test: 15, 50, 150, 500, 1500 and 5000 µg / plate
main test: 15, 50, 150, 500, 1500 and 5000 µg / plate

Vehicle / solvent:

- Vehicle(s) / solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent / vehicle: The test material was immiscible in dimethyl sulphoxide, acetone and dimethyl formamide at 50 mg/mL but was miscible in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. Tetrahydrofuran is an acceptable solvent vehicle for use in the Ames Test (6).

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrafuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test material appearance in solution was clear and colourless and dosed very well onto Vogel Bonner agar plates.

DURATION
- Preincubation period: The plates were incubated for a nominal 48 hours at 37.°C after an initial overnight equilibration period and then assessed for revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs

SELECTION AGENT (mutation assays): Not applicable.

NUMBER OF REPLICATIONS: Triplicate plating.

NUMBER OF CELLS EVALUATED: Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

OTHER EXAMINATIONS: None

Evaluation criteria:

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x E9 bacteria per ml.
All positive control chemicals must induce positive responses within the historical range for the dose level used, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria
The test material may be considered positive in this test system if the following criteria are met:
Biological relevance of the results will be considered first. Statistical methods, as recommended by the UKEMS (5) will also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. For a test material to be considered positive in this test system it should normally have induced at least a twofold increase in revertant colony frequency for TA98, T100 and WP2uvrA- and a threefold increase in TA1535 and TA1537. The effect must be reproduced in an independent assay with evidence of a dose-related response.

Statistics:

Standard deviation

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was immiscible in distilled water in solubility checks performed in-house.
- Precipitation: An oily precipitate was observed at and above 500 µg / plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation, the amino acid supplemented top agar and the S9-mix used in this experiment were shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was shown to be satisfactory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 500 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for the Main test are presented in the tables below:

Test Results: Experiment 2 (Main Test) – Without Metabolic Activation

Test Period		From: 28 March 2006					To: 31 March 2006
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	115 128 118	(120) 6.8#	20 20 30	(23) 5.8	20 20 37		(26) 9.8	14 18 25	(19) 5.6	7 9 10	(9) 1.5
-	15	120 108 104	(111) 8.3	23 35 26	(28) 6.2	32 33 26		(30) 3.8	11 25 19	(18) 7.0	8 8 10	(9) 1.2
-	50	152 103 98	(118) 29.8	26 26 16	(23) 5.8	21 26 26		(24) 2.9	19 18 16	(18) 1.5	10 8 8	(9) 1.2
-	150	76 131 121	(109) 29.3	11 13 13	(12) 1.2	26 23 23		(24) 1.7	18 18 13	(16) 2.9	4 10 10	(8) 3.5
-	500	97 P 111 P 106 P	(105) 7.1	16 P 11 P 25 P	(17) 7.1	15 P 24 P 21 P		(20) 4.6	19 P 19 P 11 P	(16) 4.6	7 P 7 P 8 P	(7) 0.6
-	1500	75 P 74 P 117 P	(89) 24.5	14 P 20 P 20 P	(18) 3.5	33 P 34 P 29 P		(32) 2.6	11 P 13 P 14 P	(13) 1.5	5 P 8 P 3 P	(5) 2.5
-	5000	115 P 90 P 91 P	(99) 14.2	19 P 23 P 20 P	(21) 2.1	20 P 21 P 40 P		(27) 11.3	16 P 18 P 15 P	(16) 1.5	7 P 7 P 8 P	(7) 0.6
Positive controls   S9-Mix   -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		542 479 527	(516) 32.9	245 235 276	(252) 21.4	694 800 878		(791) 92.4	214 206 211	(210) 4.0	776 772 800	(783) 15.1

Vehicle and dosing volume used in the test: tetrahydrofuran at 25 µl

Test Results: Experiment 2 (Main Test) – With Metabolic Activation

Test Period		From: 28 March 2006						To: 31 March 2006
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	74 89 87	(83) 8.1#	13 10 15	(13) 2.5	33 33 25	(30) 4.6		29 20 16	(22) 6.7	14 20 15	(16) 3.2
+	15	82 97 85	(88) 7.9	15 15 12	(14) 1.7	32 31 31	(31) 0.6		23 20 20	(21) 1.7	15 15 15	(15) 0.0
+	50	95 96 97	(96) 1.0	8 14 14	(12) 3.5	23 22 38	(28) 9.0		8 16 22	(15) 7.0	16 16 15	(16) 0.6
+	150	85 73 60	(73) 12.5	7 13 13	(11) 3.5	35 25 25	(28) 5.8		16 13 14	(14) 1.5	21 16 16	(18) 2.9
+	500	90 P 91 P 84 P	(88) 3.8	13 P 5 P 7 P	(8) 4.2	27 P 42 P 41 P	(37) 8.4		15 P 21 P 16 P	(17) 3.2	14 P 13 P 14 P	(14) 0.6
+	1500	86 P 85 P 85 P	(85) 0.6	21 P 9 P 9 P	(13) 6.9	31 P 27 P 27 P	(28) 2.3		13 P 13 P 24 P	(17) 6.4	14 P 18 P 15 P	(16) 2.1
+	5000	86 P 71 P 75 P	(77) 7.8	11 P 12 P 11 P	(11) 0.6	26 P 20 P 26 P	(24) 3.5		16 P 24 P 24 P	(21) 4.6	11 P 18 P 18 P	(16) 4.0
Positive controls   S9-Mix   +	Name Concentration (µg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1985 1698 1867	(1850) 144.3	167 213 203	(194) 24.2	956 1093 1076	(1042) 74.7		170 196 165	(177) 16.6	586 650 525	(587) 62.5

 

Vehicle and dosing volume used in the test: tetrahydrofuran at 25 µl

Conclusions:

Interpretation of results (migrated information): negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. The method has been designed to comply with the requirements of the Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries. The method also complies with the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC, and the USA, EPA (TSCA) OPPTS harmonised guidelines (870.5100, Aug 1998).

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichiacoli strain WP2uvrA^- were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity test and was 15 to 5000 µg / plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results. The vehicle (tetrahydrofuran) and untreated control plates produced counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg / plate. An oily precipitate was observed at and above 500 µg / plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion. The test material was considered to be non-mutagenic under the conditions of this test.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2006 to 30 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals (2006) No. 487, Draft Proposal for a New Guideline 487: In Vitro Micronucleus Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30 August 2005. Date of certificate: November 2005

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/ß-naphthoflavone-induced rat liver homogenate

Test concentrations with justification for top dose:

Experiment 1:

Group Final concentration (µg/ml)
4(16)-hour without S9 312.5, 625, 1250, 2500
4(16)-hour with S9 312.5, 625, 1250, 2500

Experiment 2

Group Final concentration (µg/ml)
4(16)-hour without S9 78.13, 156.25, 312.5, 625, 1250, 2500
4(16)-hour with S9 78.13, 156.25, 312.5, 625, 1250, 2500

Vehicle / solvent:

- Vehicle(s) / solvent(s) used: acetone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (Eagle's minimal esential medium with HEPES bufer (MEM) supplemented in-house with L-glutamine, pemicillin / streptomycin, amphotericin B and 15 % foetal calf-serum

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours +/- S9; 20 hours -S9
- Expression time (cells in growth medium): 44 hours+/-S9; 2 hours-S9
- Selection time (if incubation with a selection agent): 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours +/-S9

SELECTION AGENT (mutation assays): Cytochalasin B
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Minimum of 500 cells per culture for CBPI estimation; 2000 cells for presence of micronuclei / dose

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)

Evaluation criteria:

The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

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.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analysis may be used if appropriate, Hoffman et al (2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with aberrations which was reproducible.

The test material did not induce any statistically significant increases in the frequency of cells with micronuclei in either the absence or presence of a metabolising system, in either of two separate experiments.

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 osmolality did not increase by more than 50 mOsm.
- Precipitation: A cloudy precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure at and above 19.53 µg/ml in the 4-hour and 20-hour exposure groups in the absence of metabolic activation, and at and above 78.13 µg/ml in the 4-hour exposure group in the presence of metabolic activation. The precipitate was observed to form a greasy/oily layer at and above 156.25 µg/ml in the 4-hour exposure groups and at and above 625 µg/ml in the 20-hour exposure group.
- Other confounding effects: The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/ml. The maximum dose was based on the maximum practical dose level.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/ml. The maximum dose was based on the maximum practical dose level. A cloudy precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure at and above 19.53 µg/ml in the 4-hour and 20-hour exposure groups in the absence of metabolic activation, and at and above 78.13 µg/ml in the 4-hour exposure group in the presence of metabolic activation. The precipitate was observed to form a greasy/oily layer at and above 156.25 µg/ml in the 4-hour exposure groups and at and above 625 µg/ml in the 20-hour exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2500 µg/ml in all three of the exposure groups. The test material induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level was based on the maximum practical dose of 2500 µg/ml in all exposure groups for both experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY: There was no dose-related inhibition of CBPI observed in either the absence or presence of S9. The maximum dose level selected for binucleate cell analysis was the maximum dose level investigated (2500 µg/ml).

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Results:
All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control materials induced statistically significant increases in the frequency of cells with micronuclei, indicating the satisfactory performance of the test and of the activity of the metabolising system.
The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included the maximum practical dose level.
Conclusion:
The test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

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

Reference 4

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:

10 May 2006 - 15 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD method 487 (draft)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable (chromosome aberration test)

Species / strain / cell type:

lymphocytes: Human lymphocytes

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
suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a
viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine)
incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

PB/BetaNF S9

Test concentrations with justification for top dose:

I. Preliminary toxicity test:
Dose range 19.5 to 5000 µg/mL.

II. Micronucleus Test - Experiment 1:
4(16)-hour without S9: 0*, 39, 78.1, 156.25, 312.5*, 625*, 1250*, MMC 0.2* [µg/mL];
4(16)-hour with S9: 0*, 39, 78.1, 156.25, 312.5*, 625*, 1250*, CP 5* [µg/mL].

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

III. Micronucleus Test - Experiment 2:
20-hour without S9: 0*, 39, 78.1, 156.25, 312.5*, 625*, 1250*, DC 0.075* [µg/mL];
4(16)-hour with S9: 0*, 39, 78.1, 156.25, 312.5*, 625*, 1250*, CP 5* [µg/mL]

* = Dose levels selected for binucleate analysis
DC = Demecolcine
CP = Cyclophosphamide

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Experiment 1: Positive control material in the absence of S9 [mitomycin C (MMC)]: HEPES buffer (MEM)
- Experiment 2: Positive control material in the absence of S9 [demecolcine (DC)]: Water
- Experiment 1 & 2: Positive control material in the presence of S9 [cyclophosphamide (CP)]: DMSO
- Justification for choice of solvent/vehicle: test material is not soluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into
sterile plastic flasks for each culture:
9.05 mL MEM, 15% (FCS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinised whole blood

DURATION
- Preincubation period: Not reported.
- Exposure duration:
I. With Metabolic Activation (S9) Treatment:
After approximately 48 hours incubation at 37°C with 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged.
Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control; test material or positive control was added to each culture. 1 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the
incubator. The nominal final volume of each culture was 10 mL.
After 4 hours, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated
for a further 16 hours before the addition of Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 28 hours.

II. Without Metabolic Activation (S9) Treatment:
In Experiment 1, after approximately 48 hours incubation at 37ºC with 5% CO2 in humidified air the cultures were decanted into tubes and
centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test material solution or positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at 37ºC, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture
medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells
were then returned to the incubator for a further 16 hours before the addition of Cytochalasin B, at a final concentration of 4.5 μg/mL, and then
incubated for a further 28 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 20 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test material dose solution or positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated for 20 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 28 hours.


NUMBER OF REPLICATIONS:
Two replicates (A and B).

NUMBER OF CELLS EVALUATED:
CBPI: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, bi-nucleate and multinucleate cells and the
CBPI value expressed as a percentage of the vehicle controls.
Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture,
two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation and a continuous
exposure of 20 hours without metabolic activation. In all exposure groups Cytochalasin B was added 20 hours after the initiation of exposure and
the cells were harvested 48 hours after the initiation of exposure. The dose range of test material used was 19.5 to 5000 μg/mL. Parallel flasks,
containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations
could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected
for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test material toxicity and for selection of the dose levels for the experiments of the main study.

Evaluation criteria:

- Qualitative Slide Assessment: The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and
extent of precipitation, if any, of the test material. These observations were used to select the dose levels for CBPI evaluation.

- Cytokinesis Block Proliferation Index (CBPI): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate,
bi-nucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.

- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture,
two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.
Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer
donors.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on
observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical
analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with aberrations, which was reproducible.

Species / strain:

lymphocytes: Human 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change of pH when the test material was dosed into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: None
- Precipitation: Observations in the preliminary toxicity test: A cloudy precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure at and above 78.1 μg/mL, in both exposure groups without metabolic activation. The precipitate was observed to form a
greasy oily layer at and above 625 μg/mL, and it was considered that maximumexposure to the cells was occurring at about 1250 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present in all dose levels up to 5000
μg/mL in all three of the exposure groups.
The selection of the maximum dose level was based on the onset of the greasy oily precipitate and was limited to 1250 μg/mL in all exposure groups
for both experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material induced no evidence of toxicity in any of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Results of Micronucleus Test - Experiment 1 Without S9

Concentration μg/mL	Exposure Time +/-S9	Replicate	CBPI	% Control CBPI	Micronuclei (MN) Per % Control 1000 Bi-nucleate cells			% Cells with MN	Mean % Cells with MN
					1 MN	2 MN	>2 MN
0	4 hr, -S9	A B	2.29 2.27	100	7 8	0 0	1 0	0.80 0.80	0.80
312.5		A B	2.22 2.26	97	6 6	1 2	0 0	0.70 0.80	0.75
625		A B	2.27 2.37	103	8 5	2 0	0 0	1.00 0.50	0.75
1250		A B	2.24 2.28	98	11 7	2 0	0 0	1.30 0.70	1.00
MMC 0.2		A B	2.25 2.22	96	56 54	4 0	0 1	6.00 5.50	5.75***

MMC = Mitomycin C; *** = P < 0.001

 

Table 2: Results of Micronucleus Test - Experiment 1 With S9

Concentration μg/mL	Exposure Time +/-S9	Replicate	CBPI	% Control CBPI	Micronuclei (MN) Per % Control 1000 Bi-nucleate cells			% Cells with MN	Mean % Cells with MN
					1 MN	2 MN	>2 MN
0	4 hr, +S9	A B	2.39 2.31	100	9 10	0 1	0 0	0.90 1.10	1.00
312.5		A B	2.31 2.33	98	10 7	1 0	0 1	1.10 0.80	0.95
625		A B	2.35 2.39	101	8 5	1 0	1 0	1.00 0.50	0.75
1250		A B	2.32 2.36	99	6 8	1 1	0 0	0.70 0.90	0.80
CP 5.0		A B	2.23 2.29	93	57 59	13 7	3 3	7.30 6.90	7.10***

CP = Cyclophosphamide; *** = P < 0.001

 

 

Table 3: Results of Micronucleus Test - Experiment 2 Without S9

Concentration μg/mL	Exposure Time +/-S9	Replicate	CBPI	% Control CBPI	Micronuclei (MN) Per % Control 1000 Bi-nucleate cells			% Cells with MN	Mean % Cells with MN
					1 MN	2 MN	>2 MN
0	20 hr, -S9	A B	1.99 1.98	100	6 9	2 0	0 0	0.80 0.90	0.85
312.5		A B	1.91 1.99	96	14 8	0 1	0 0	1.40 0.90	1.15
625		A B	1.97 2.06	103	7 10	0 0	1 1	0.80 1.10	0.95
1250		A B	2.03 2.08	107	13 9	0 0	1 0	1.40 0.90	1.15
DC 0.075		A B	1.67 1.62	65	31 36	3 5	1 2	3.50 4.30	3.90***

DC = Demecolcine; *** = P < 0.001

 

 

Table 4: Results of Micronucleus Test - Experiment 2 With S9

Concentration μg/mL	Exposure Time +/-S9	Replicate	CBPI	% Control CBPI	Micronuclei (MN) Per % Control 1000 Bi-nucleate cells			% Cells with MN	Mean % Cells with MN
					1 MN	2 MN	>2 MN
0	4 hr, +S9	A B	2.08 2.09	100	10 7	1 1	0 0	1.10 0.80	0.95
312.5		A B	2.07 2.14	102	9 9	1 1	0 0	1.00 1.00	1.00
625		A B	2.11 2.15	104	11 7	1 0	0 0	1.20 0.70	0.95
1250		A B	2.11 2.13	103	8 7	1 0	0 0	0.90 0.70	0.80
CP 5.0		A B	1.84 1.83	77	69 69	4 6	2 1	7.50 7.60	7.55***

CP = Cyclophosphamide; *** = P < 0.001

Conclusions:

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

Interpretation of results: negative (non-clastogenic and non-aneugenic)

Executive summary:

This report describes the results of an in vitro study for the detection of structural and numerical chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations. The method used followed that described in the OECD Guidelines for Testing of Chemicals (2006) No. 487 "Draft Proposal for a New Guideline 487: In Vitro Micronucleus Test", but has been modified to more closely resemble the OECD 473 guideline for the chromosome aberration test.

All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control materials induced statistically significant increases in the frequency of cells with micronuclei, indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included the lowest moderately precipitating dose level. The test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 5

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:

2014-07-21 to 2014-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C15 to C19

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:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Experiment II: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL

Without metabolic activation:
Experiment I: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Experiment II: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluatedAt least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

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 applicable

Positive controls validity:

valid

Additional information on results:

The test item Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2400.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. Phase separation was observed at the end of treatment in Experiment I and II in the presence of S9 mix at 83.6 µg/mL and above. In the absence of S9 mix phase separation was observed in Experiment I at 146.2 µg/mL and above and in Experiment II at 15.6 µg/mL and above.
No relevant influence on osmolarity or pH value was observed.
No relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration (Table 3 - Table 4).
Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, one single statistically significant increase was observed after treatment with 783.7 µg/mL (3.0 % aberrant cells, excluding gaps) (Table 7). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of S9 mix, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 µg/mL (Table 10). Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 or 770.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Table2     Summary of results of the chromosomal aberration study with      
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics    

Exp.	Preparationinterval	Test itemconcentration in µg/mL	Mitotic indices in % of control	Aberrant cells in %
				incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	100.0	2.5	2.5	0.5
		Positive control^2	120.8	10.0	10.0^S	2.0
		83.6	102.9	2.0	2.0	0.0
		783.7^PS	88.2	2.5	2.5	0.0
		1371.4^PS	91.8	3.0	3.0	0.0
		2400.0^PS	124.5	2.5	2.0	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^3#	31.9	40.0	39.0^S	12.0
		783.7^PS	106.1	2.5	1.5	0.0
		1371.4^PS	105.2	1.0	1.0	0.0
		2400.0^PS	94.8	2.0	1.5	0.0

*   Including cells carrying exchanges

^#    Evaluation of 50 metaphases per culture

^##  Evaluation of 200 metaphases per culture

^PS  Phase separation occurred at the end of treatment

^S    Aberration frequency statistically significant higher than corresponding control values

¹    Acetone         0.5 % (v/v)

²     EMS          770.0 µg/mL

³     EMS          660.0 µg/mL

Table 2, cont.  Summary of results of the chromosomal aberration study with           
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics         

Exp.	Preparationinterval	Test itemconcentration in µg/mL	Mitotic indices in % of control	Aberrant cells in %
				incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^2	31.6	9.5	9.5^S	1.5
		47.7	72.6	1.0	1.0	0.0
		783.7^PS	77.7	3.0	3.0^S	0.0
		1371.4^PS	88.9	0.0	0.0	0.0
		2400.0^PS	95.2	1.5	1.5	0.0
II	22 hrs	Solvent control^1	100.0	1.5	1.5	0.0
		Positive control^3	35.5	18.0	18.0^S	3.0
		27.3	107.6	1.0	0.5	0.0
		47.7^##	112.7	4.3	3.8	0.3
		83.6^PS	104.2	3.0	2.5	0.0
		783.7^PS	110.9	1.0	1.0	0.0
		1371.4^PS	116.4	1.5	1.5	0.0
		2400.0^PS	113.6	2.5	1.5	0.5

*   Including cells carrying exchanges

^#    Evaluation of 50 metaphases per culture

^##  Evaluation of 200 metaphases per culture

^PS  Phase separation occurred at the end of treatment

^S    Aberration frequency statistically significant higher than corresponding control values

¹    Acetone         0.5 % (v/v)

²    CPA               2.5 µg/mL

³    CPA               7.5 µg/mL

Conclusions:

A chromosome aberration study was performed for Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics according to OECD 473 and GLP. In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration (2400 μg/mL). Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of metabolic activation, one single statistically significant increase was observed after treatment with 783.7 μg/mL (3.0 % aberrant cells, excluding gaps). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of metabolic activation, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 μg/mL. Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Executive summary:

The test item Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2400.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data inaccordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, one single statistically significant increase was observed after treatment with 783.7 µg/mL (3.0 % aberrant cells, excluding gaps). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of S9 mix, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 µg/mL. Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study (cytogenicity / bone marrow chromosome aberration) on closely related substances 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Distillates (Fischer-Tropsch), C8-C26, branched and linear': negative (OECD 475)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow 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: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

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.

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:

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.

Interpretation of results: negative

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.

Reference 2

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: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

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:

Sufficient Wistar HanTM (RCCHanTMWIST) rats were supplied by a reputable supplier. For full details please see full study report. At the start of the test the rats weighed 186 to 229 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 by sex in solid-floor polypropylene cages with woodflake bedding (Supplied by a reputable supplier. For full details please see full study report). 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 (Supplied by a reputable supplier. For full details please seefull study report).

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:

The vehicle control was supplied by a reputable supplier as follows:
Supplier's identification: Arachis oil
Harlan serial number: 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 (see in attached section). 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.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mortality Data and Clinical Observations
There were no premature deaths seen in any of the test item dose groups and no clinical signs were observed in animals dosed with the test item at any of the dose levels.

Evaluation of Bone Marrow Slides
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 he attached Tables 2 to 7. (See in attached section).

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 (see table 2 in the attached section).

The positive control group animals showed highly significant increases in the frequency of aberrations (see table 3 in the attached section) 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.

Table 1: Group Mean Results of Chromosome Aberration Test

Table 2: Individual Results of Chromosome Aberration Test - 24-Hour Vehicle Control Group

Table 3: Individual Results of Chromosome Aberration Test - 24-Hour Positive Control Group: Cyclophosphamide 25 mg/kg

Table 4: Individual Results of Chromosome Aberration Test - 48-Hour Test Item Group: 2000 mg/kg

Table 5: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 2000 mg/kg

Table 6: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 1000 mg/kg

Table 7: Individual Results of Chromosome Aberration Test - 24-Hour Test Item Group: 500 mg/kg

Conclusions:

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.

Interpretation of results: negative

Executive summary:

Introduction. 

The study was performed to assess the potential of the test item to produce damage to chromosomes or the mitotic apparatus when administered to rats.   The method used is compatible with that described in the revised OECD Guidelines for Testing of Chemicals No. 475 “Mammalian Bone MarrowChromosome 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 Safepharm Laboratories Ltd*(Project No 2041/0045) 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 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.

*On the 9^thNovember 2008 Safepharm Laboratories Ltd changed its name to Harlan Laboratories Ltd.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item 'Paraffin Waxes (Fischer-Tropsch), full range, C15-50 - branched 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 test material was considered to be non-mutagenic under the conditions of this test.

An in vitro micronucleus study has been conducted using the test item 'Paraffin Waxes (Fischer-Tropsch), full range, C15-50 - branched 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. 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 can be obtained from OECD 473 guideline studies of the related substances 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Distillates (Fischer-Tropsch), C8-C26, branched and linear' and ' Hydrocarbons, C15-C19, n-alkanes, isoalkanes, < 2% aromatics (EC 940-730-5).

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 in vivo chromosome aberration studies on the substances ' Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Distillates (Fischer-Tropsch), C8-C26, branched and linear', conducted according to OECD 475:

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

Based on the available studies on the registered and the related substances it can be concluded that 'Paraffin waxes (Fischer-Tropsch), full range, C15-C50, branched and linear' is not genotoxic.

Justification for classification or non-classification

Based on the available in vitro and in vivo data the substance 'Paraffin waxes (Fischer-Tropsch), full range, C15-C50, branched and linear' is not genotoxic and does not require classification according to Regulation 1272/2008/EC.