Kerosine (Fischer-Tropsch), full-range, C8-16-branched and linear

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Safepharm Laboratories Limited

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-670-5

EC Name:

-

Cas Number:

848301-66-6

Molecular formula:

As the substance is a UVCB substance neither a molecular formular nor the molecular weight can be defined.

IUPAC Name:

C8-C16 branched and linear hydrocarbons (full range) – Kerosine

Details on test material:

- Name of test material (as cited in study report): Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear
- Substance type: Organic
- Physical state: 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

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 S9

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. The effect must be reproduced in an independant assay with evidence of a dose-related response.

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 1500 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). results are summarised in attached tables 1 and 2.


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	120 115 129	(121) 7.1#	26 20 27	(24) 3.8	25 18 18	(20) 4.0	25 32 32	(30) 4.0	10 9 9	(9) 0.6
without	15	98 87 104	(96) 8.6	25 19 21	(22) 3.1	22 21 30	(24) 4.9	26 14 18	(19) 6.1	11 7 10	(9) 2.1
without	50	130 104 133	(122) 15.9	33 24 24	(27) 5.2	20 25 19	(21) 3.2	29 26 23	(26) 3.0	13 13 9	(12) 2.3
without	150	74 91 110	(92) 18.0	27 23 29	(26) 3.1	36 24 21	(27) 7.9	40 22 22	(28) 10.4	13 10 9	(11) 2.1
without	500	110 106 113	(110) 3.5	38 19 33	(30) 9.8	22 22 20	(21) 1.2	23 19 18	(23) 5.5	15 11 14	(13) 2.1
without	1500	70P 77P 91P	(79) 10.7	22P 21P 21P	(21) 0.6	25P 27P 22P	(25) 2.5	26P 36P 26P	(29) 5.8	5P 4P 5P	(5) 0.6
without	5000	79P 70P C P	(75) 6.4	19P 27P 21P	(22) 4.2	46P 27P 22P	(32) 12.7	21P 19P 22P	(21) 1.5	10P 3P 9P	(7) 3.8
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;

C: Contaminated; 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	73 96 95	(88) 13.0#	13 18 18	(16) 2.9	29 25 27	(27) 2.0	23 31 31	(28) 4.6	12 21 11	(15) 5.5
with	15	81 100 77	(86) 12.3	14 8 11	(11) 3.0	23 30 33	(29) 5.1	18 34 29	(27) 8.2	12 15 22	(16) 5.1
with	50	88 81 89	(86) 4.4	27 12 18	(19) 7.5	33 34 16	(28) 10.1	C 26 27	(27) 0.7	18 14 21	(18) 3.5
with	150	75 90 65	(77) 12.6	8 11 10	(10) 1.5	29 23 23	(25) 3.5	24 26 26	(25) 1.2	14 14 7	(12) 4.0
with	500	54 74 73	(67) 11.3	22 10 5	(12) 8.7	38 33 29	(33) 4.5	32 26 24	(27) 4.2	14 14 15	(14) 0.6
with	1500	70P 74P 70P	(71) 2.3	12P 10P 13P	(12) 1.5	25P 33P 27P	(28) 4.2	29P 29P 35P	(31) 3.5	10P 21P 14P	(15) 5.6
with	5000	69P 59P 62P	(63) 5.1	11P 11P 7P	(10) 2.3	34P 31P 35P	(33) 2.1	37P 33P 31P	(34) 3.1	18P 12P 13P	(14) 3.2
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	87 77 119	(94) 21.9#	17 13 17	(16) 2.3	22 23 18	(21) 2.6	29 24 22	(25) 3.6	17 20 17	(18) 1.7
without	15	79 89 80	(83) 5.5	11 20 28	(20) 8.5	32 27 25	(28) 3.6	31 19 16	(22) 7.9	9 14 11	(11) 2.5
without	50	82 90 72	(81) 9.0	18 11 25	(18) 7.0	18 21 20	(20) 1.5	14 27 20	(20) 6.5	14 11 11	(12) 1.7
without	150	75 94 70	(80) 12.7	11 17 18	(15) 3.8	34 17 19	(23) 9.3	19 27 22	(23) 4.0	11 16 11	(13) 2.9
without	500	68 85 68	(74) 9.8	17 23 19	(20) 3.1	30 20 26	(25) 5.0	28 17 17	(21) 6.4	10 15 9	(11) 3.2
without	1500	72P 74P 76P	(74) 2.0	18P 20P 10P	(16) 5.3	27P 29P 22P	(26) 3.6	25P 24P 21P	(23) 2.1	4P 18P 13P	(12) 7.1
without	5000	74P 63P 67P	(70) 3.8	16P 6P 27P	(16) 10.5	25P 24P 26P	(25) 1.0	24P 23P 20P	(22) 2.1	10P 8P 10P	(9) 1.2
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	(210) 4.0	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	91 112 103	(102) 10.5#	14 16 21	(17) 3.6	31 40 32	(34) 4.9	17 20 20	(19) 1.7	7 11 11	(10) 2.3
with	15	68 89 99	(85) 15.8	11 15 10	(12) 2.6	28 29 26	(28) 1.5	41 13 16	(23) 15.4	10 14 11	(12) 2.1
with	50	85 70 108	(88) 19.1	6 10 16	(11) 5.0	42 25 24	(30) 10.1	18 18 25	(20) 4.0	16 6 14	(12) 5.3
with	150	85 79 113	(92) 18.1	6 13 25	(15) 9.6	21 31 37	(30) 8.1	22 18 21	(20) 2.1	16 19 9	(15) 5.1
with	500	112 80 79	(90) 18.8	13 9 10	(11) 2.1	24 38 31	(31) 7.0	14 20 18	(17) 3.1	14 11 14	(13) 1.7
with	1500	103P 119P 108P	(110) 8.2	8P 8P 7P	(8) 0.6	23P 26P 29P	(26) 3.0	19P 33P 17P	(23) 8.7	9P 14P 25P	(16) 8.2
with	5000	79P 85P 76P	(80) 4.6	10P 11P 13P	(11) 1.5	31P 30P 24P	(28) 3.8	24P 22P 18P	(21) 3.1	14P 17P 14P	(15) 1.7
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 with metabolic activation
negative without metabolic activation

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 'Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear' was found to be non-mutagenic under the conditions of this test.

Executive summary:

The test substance 'Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear' 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. Kerosine (Fischer-Tropsch), full range, C8-16 - branched and linear was considered to be non-mutagenic under the conditions of this test.