C8-C26 branched and linear hydrocarbons – Distillates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-03-07 to 2006-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-740-5

EC Name:

-

Cas Number:

848301-67-7

Molecular formula:

main general molecular formula: CnH(2n+2)

IUPAC Name:

2,2,3,4,4,5,6-heptamethylheptane; 2,2,8-trimethyltetradecane; 2,2-dimethyldodecane; 2,2-dimethyltetradecane; 2-methyltetradecane; 3,5,7-triethyldecane; 4-ethyldodecane; 4-propyldodecane; 5,5-dipropylnonane; 5,7-dimethyl-4-propylundecane; 5-butyl-5-propyldecane; 5-butyl-5-propylnonane; 5-ethyl-5-propylnonane; 5-ethyl-7-methyl-4-propylundecane; 5-propyltridecane; 6-butyl-6-methyldodecane; 6-butyldodecane; 6-butylundecane; 7-methyl-4-propylundecane; 8-methyl-5-propyltridecane; 9-butyl-6-propylhexadecane; heptadecane; hexacosane; hexadecane; octane; pentadecane; tetradecane

Details on test material:

- Name of test material (as cited in study report): Distillates (Fischer-Tropsch), C8-26 branched and linear
- Substance type: Organic
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): Information not available
- Composition of test material, percentage of components: Information not available
- Isomers composition: Information not available
- Purity test date: Information not available
- Lot/batch No.: Information not available
- Expiration date of the lot/batch: Information not available
- Stability under test conditions: Stable under normal temperature and pressure
- Storage condition of test material: Room temperature, in the dark

Method

Target gene:

histidine auxotrophs of Salmonella typhimurium (have also a deletion through the excision repair gene (uvrB-) and tryptophan auxotrophs of Escherichia coli (has also a deletion in an excision repair gene uvrA-)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

S9 cells from livers of Spraque-Dawley rats

Additional strain / cell type characteristics:

DNA polymerase A deficient

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

S9 cells from livers of Spraque-Dawley rats

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-naphtoflavone induced, rat-liver S915-5000

Test concentrations with justification for top dose:

0, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test substance not soluble in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: further control substances: 9AA, ENNG

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Minimal / In suspension; 0.1 mL of the bacterial culture was mixed with 0.1 mL test material,
0.5 mL of phosphate buffer or S9-mix and 2 mL of molten, trace histidine or tryptophsan supplemented,
top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar.
DURATION
- Preincubation period: Preculture period lasted 10 hours.
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
The plates were incubated for 48 h 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.

Evaluation criteria:

Sensitivity of the essay and the efficacy of the S9-mix were validated.

Statistics:

Statistical methods are 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, TA100 and WP2uvrA- and a threefold increase in TA1535 and TA1537.

Results and discussion

Test results

Test results 1

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

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: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: None
- Precipitation: An oily precipitate was observed at and above 500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
Range-finding test was performed (experiment 1)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was not relevant at the concentration range tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test results: Experiment 1 (Range-finding test) – Without Metabolic activation

Test period		Number of revertants (mean number of colonies per plate
With or without S9-Mix	Test substance concentration (µg/plate)	Base-pair substitution type						Frameshift type
		TA 100		TA1535		WP2uvtA-		TA98		TA1537
without	0	118 97 103	(106) 10.8#	34 19 26	(26) 7.5	20 12 33	(22) 10.6	36 35 48	(40) 7.2	13 4 8	(8) 4.5
without	15	101 99 101	(100) 1.2	21 14 20	(18) 3.8	18 22 22	(21) 2.3	38 35 27	(33) 5.7	14 11 10	(12) 2.1
without	50	100 86 91	(92) 7.1	34 25 24	(28) 5.5	26 20 21	(22) 3.2	30 30 49	(36) 11.0	20 5 13	(13) 7.5
without	150	89 106 96	(97) 8.5	20 25 21	(22) 2.6	20 38 24	(27) 9.5	23 21 16	(20) 3.6	5 10 4	(6) 3.2
without	500	103P 84P 88P	(92) 10.0	24P 22P 21P	(22) 1.5	14P 27P 25P	(22) 7.0	24P 16P 23P	(21) 4.4	7P 7P 9P	(8) 1.2
without	1500	103P 84P 88P	(102) 9.2	18P 23P 23P	(21) 2.9	18P 14P 25P	(19) 5.6	23P 26P 25P	(25) 1.5	3P 3P 2P	(3) 0.6
without	5000	103P 84P 88P	(96) 2.6	26P 29P 21P	(25) 4.0	15P 23P 21P	(20) 4.2	32P 25P 45P	(34) 10.1	3P 7P 9P	(6) 3.1
Positive controls	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Concentration (µg/plate)	3		5		2		0.2		80
Without S9	Number of colonies per plate	621 662 670	(651) 26.3	278 267 227	(257) 26.8	806 839 848	(831) 22.1	189 187 132	(169) 32.3	1525 835 1057	(1139) 352.2

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine; P: Precipitate; #: Standard Deviation

 

Table 2: Test results: Experiment 1 (Range-finding test) – With Metabolic activation

Test period		Number of revertants (mean number of colonies per plate
With or without S9-Mix	Test substance concentration (µg/plate)	Base-pair substitution type						Frameshift type
		TA 100		TA1535		WP2uvtA-		TA98		TA1537
with	0	89 87 93	(90) 3.1#	16 12 18	(15) 3.1	37 47 38	(41) 5.5	44 37 34	(38) 5.1	16 16 20	(17) 2.3
with	15	87 101 95	(94) 7.0	13 14 16	(14) 1.5	26 35 30	(30) 4.5	26 22 33	(27) 5.6	21 25 12	(19) 6.7
with	50	87 86 97	(90) 6.1	18 14 13	(15) 2.6	33 19 34	(29) 8.4	22 24 38	(28) 8.7	9 11 16	(12) 3.6
with	150	100 80 103	(94) 12.5	9 14 15	(13) 3.2	29 31 25	(28) 3.1	29 31 27	(29) 2.0	15 13 13	(14) 1.2
with	500	90P 91P 99P	(93) 4.9	11P 22P 15P	(16) 5.6	29P 32P 41P	(34) 6.2	16P C P 30P	(23) 9.9	13P 12P 15P	(13) 1.5
with	1500	107P 103P 104P	(105) 2.1	12P 15P 11P	(13) 2.1	23P 38P 44P	(35) 10.8	37P 37P 26P	(33) 6.4	18P 15P 4P	(12) 7.4
with	5000	100P 97P 84P	(94) 8.5	8P 16P 14P	(13) 4.2	32P 31P 34P	(32) 1.5	35P 38P 32P	(35) 3.0	13P 15P 10P	(13) 2.5
Positive controls	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
With S9	Number of colonies per plate	2142 2096 2220	(2153) 62.7	240 224 252	(239) 14.0	496 464 539	(500) 37.6	212 213 203	(209) 5.5	636 615 643	(631) 14.6

2AA: 2-Aminoanthracene; BP: Bonzo(a)pyrene, C: Contaminated, P: Precipitate; #: Standard Deviation

 

Table 3: Test results: Experiment 2 (Main test) – Without Metabolic activation

Test period		Number of revertants (mean number of colonies per plate
With or without S9-Mix	Test substance concentration (µg/plate)	Base-pair substitution type						Frameshift type
		TA 100		TA1535		WP2uvtA-		TA98		TA1537
without	0	106 95 91	(97) 7.8#	15 20 21	(19) 3.2	18 15 24	(19) 4.6	22 21 24	(22) 1.5	11 7 11	(10) 2.3
without	15	78 79 71	(76) 4.4	22 18 18	(19) 2.3	27 18 18	(21) 5.2	19 29 19	(22) 5.8	12 18 10	(13) 4.2
without	50	78 133 110	(107) 27.6	18 18 21	(19) 1.7	20 20 27	(22) 4.0	26 22 22	(23) 2.3	9 14 9	(11) 2.9
without	150	97 106 109	(104) 6.2	19 19 16	(18) 1.7	25 22 22	(23) 1.7	23 23 22	(23) 0.6	8 8 13	(10) 2.9
without	500	93P 111P 93P	(99) 10.4	26P 23P 30P	(26) 3.5	20P 20P 19P	(20) 0.6	22P 22P 22P	(22) 0.0	10P 10P 11P	(10) 0.6
without	1500	106P 78P 80P	(88) 15.3	12P 24P 14P	(17) 6.4	20P 13P 13P	(15) 4.0	24P 24P 21P	(23) 1.7	18P 10P 10P	(13) 4.6
without	5000	111P 93P 89P	(98) 11.7	13P 14P 8P	(12) 3.2	18P 34P 23P	(25) 8.2	22P 18P 22P	(21) 2.3	4P 4P 9P	(6) 2.9
Positive controls	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Concentration (µg/plate)	3		5		2		0.2		80
Without S9	Number of colonies per plate	542 479 527	(516) 32.9	245 235 276	(252) 21.4	694 800 878	(791) 92.4	214 206 211	(169) 32.3	776 772 800	(783) 15.1

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine; P: Precipitate; #: Standard Deviation

 

Table 4: Test results: Experiment 2 (Main test) – With Metabolic activation

Test period		Number of revertants (mean number of colonies per plate
With or without S9-Mix	Test substance concentration (µg/plate)	Base-pair substitution type						Frameshift type
		TA 100		TA1535		WP2uvtA-		TA98		TA1537
with	0	82 82 86	(77) 9.2#	11 12 13	(12) 1.0	41 38 36	(38) 2.5	27 24 33	(28) 4.6	20 16 12	(16) 4.0
with	15	71 78 78	(76) 4.0	12 12 18	(14) 3.5	35 44 35	(38) 5.2	29 29 29	(29) 0.0	20 10 10	(13) 5.8
with	50	53 58 76	(62) 12.1	7 9 9	(8) 1.2	20 26 26	(24) 3.5	15 23 23	(20) 4.6	12 12 15	(13) 1.7
with	150	79 78 79	(79) 0.6	8 7 14	(10) 3.8	25 24 24	(24) 0.6	21 21 24	(22) 1.7	19 13 13	(15) 3.5
with	500	93P 97P 79P	(90) 9.5	7P 9P 15P	(10) 4.2	27P 27P 29P	(28) 1.2	24P 27P 26P	(26) 1.5	18P 18P 15P	(17) 1.7
with	1500	86P 84P 75P	(82) 5.9	10P 4P 11P	(8) 3.8	33P 36P 30P	(33) 3.0	33P 29P 34P	(32) 2.6	12P 13P 12P	(12) 0.6
with	5000	73P 60P 66P	(66) 6.5	9P 4P 5P	(6) 2.6	32P 38P 38P	(36) 3.5	36P 23P 23P	(27) 7.5	16P 12P 15P	(14) 2.1
Positive controls	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
With S9	Number of colonies per plate	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

2AA: 2-Aminoanthracene; BP: Bonzo(a)pyrene, P: Precipitate; #: Standard Deviation

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges. No statistically significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the substance, either with or without metabolic activation. The substance 'Distillates (Fischer-Tropsch), C8-26, branched and linear' was found to be non-mutagenic under the conditions of this test.

Executive summary:

Test substance was investigated for possible genotoxic effects in an in-vitro study according to OECD guideline 471 as well as US-EPA TSCA OPPTS guideline 870.5100. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated at six dose levels (15 to 5000 µg/plate). The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of this test.