Naphtha (Fischer-Tropsch), light, C4-10-branched and linear

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- OECD 471: negative

- OECD 490 (on supporting substance with limited range, C7-C10): negative

- OECD 487 (Draft): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli

Species / strain / cell type:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

TA 100 (without S9): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate
TA 100 (with S9): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA1535 (with and without S9), TA98 and TA1537 (without S9): 5 to 5000 µg/plate
WP2 uvr A (with and without S9), TA98 and TA1537 (with S9): 15 to 5000 µg/plate

Vehicle / solvent:

acetone

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

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' was found to be non-mutagenic under the conditions of this test.

Interpretation of results: negative.

Executive summary:

According to OECD guideline 471, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA" were treated with the test material using the Ames plate incorporation method at up to seven 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 ranged between 1.5 and 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using an amended dose range from Experiment 1 (0.5 to 5000 ug/plate), fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels were included to allow for test material induced toxicity to ensure that a minimum of four non-toxic dose levels were plated out

The test material caused a visible reduction in the growth of the bacterial background lawn initially at 500 ug/plate to all Salmonella tester strains (with and without metabolic activation), predominately the base-pair substitution strains. No toxicity was observed to the Ecoli strain WP2uvrA". The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A slight oily precipitate was observed at 5000 ug/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.

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2006 - 12 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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*, 4.88, 9.75*, 19.5*, 39*, 78.1, 117.2, 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:
- Test material: 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

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 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 ofn 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

Remarks:

observed only in 20-hour exposure group

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: In the 20 hour exposure group without S9 a cloudy precipitate was observed at 312.5 μg/ml, which formed into an oily precipitate at and above 625 μg/ml. An oily precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure at and above 1250 μg/ml, in the four hour exposure groups both with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The assessment of the slides from the 20-hour without S9 exposure group indicated gross toxicity from 156.25 to 1250 μg/ml, and then apparent
recovery at 2500 and 5000 μg/ml. It was considered that this was due to the test material achieving a dose level beyond its maximum solubility in the tissue culture media and forming an oily layer on top of the culture medium. This was not observed because the test material was colourless. It was
therefore concluded that maximum exposure was achieved at 1250 μg/ml. This was confirmed by the presence of haemolysis in the blood cultures in the test material dose levels up to 1250 μg/ml, and then a reduction to no haemolysis at 2500 and 5000 μg/ml. Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 5000 μg/ml in the 4-hour exposure, both in the presence
and absence of metabolic activation (S9). The maximum dose with binucleate cells in the 20-hour continuous exposure was 5000 μg/ml, although it was noted that no binucleate cells were observed at dose levels between 156.25 and 1250 μg/ml.
The selection of the maximum dose level was based on the toxicity observed in the 20-hour exposure group and was limited to 1250 μg/ml in the 4-hour pulse exposure groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY (Preliminary Toxicity Test):
The test material induced evidence of toxicity only in the 20-hour continuous exposure group.

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	2.09 2.09	100	12 8	1 0	1 0	1.40 0.80	1.10
312.5		A B	2.07 1.95	93	15 20	1 1	1 0	1.70 2.10	1.90*
625		A B	2.10 2.09	100	18 6	1 2	0 1	1.90 0.90	1.40
1250		A B	2.03 2.07	96	14 12	7 1	1 0	2.20 1.30	1.75
MMC 0.2		A B	2.02 2.04	94	86 83	10 7	1 1	9.70 9.10	9.40***

MMC = Mitomycin C; * = P < 0.05; *** = 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	2.07 2.17	100	14 14	0 0	0 0	1.40 1.40	1.40
312.5		A B	2.13 2.06	98	17 17	1 3	0 0	1.80 2.00	1.90
625		A B	2.06 2.14	98	15 14	1 1	0 0	1.60 1.50	1.55
1250		A B	2.12 2.06	97	11 16	0 1	1 0	1.20 1.70	1.45
CP 5.0		A B	1.89 1.94	82	82 82	13 6	1 1	9.60 8.90	9.25***

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	1.96 2.04	100	7 9	3 5	1 0	1.10 1.40	1.25
9.75		A B	1.95 2.03	99	9 15	2 0	0 1	1.10 1.60	1.35
19.5		A B	2.03 2.00	102	8 10	2 0	0 0	1.00 1.00	1.00
39		A B	2.00 1.95	98	11 8	1 0	0 0	1.20 0.80	1.00
DC 0.075		A B	1.73 1.77	75	27 33	4 4	7 1	3.80 3.80	3.80***

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	2.19 2.17	100	7 8	0 0	0 1	0.70 0.90	0.80
312.5		A B	1.92 2.00	81	11 13	1 1	0 0	1.20 1.40	1.30
625		A B	2.00 2.02	86	9 11	0 0	2 0	1.10 1.10	1.10
1250		A B	2.05 2.05	89	15 11	1 0	0 1	1.60 1.20	1.40
CP 5.0		A B	2.04 1.83	79	84 95	7 14	2 2	9.30 11.10	10.20***

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.

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 toxic only in the 20-hour exposure group without metabolic activation, 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 or was limited by test material toxicity. The test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

other: L5178Y TK+/- Mouse Lymphoma Assay

Target gene:

Thymidine Kinase

Details on mammalian cell type (if applicable):

L51787 TK+/- Mouse Lymphoma cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver homogenate (S9 mix)

Test concentrations with justification for top dose:

The non-activated cultures were treated with a range of concentrations from 0.08 µl/ml to 0.005 µl/ml. The S-9 activated cultures that were cloned were treated with a range of concentrations from 0.8 µl/ml to 0.00004 µl/ml. After a two day expression period, eight non-activated and nine S-9
activated cultures were selected for cloning based on their degree of toxicity. The non-activated cultures that were cloned were treated with 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, 0.01 or 0.005 µl/ml. These concentrations produced a range in Suspension Growth of 24% to 109%.

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The results of the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of these tests, Naphtha (petroleum), light alkylate (EC 265-068-8) produced a negative response in the presence and absence of exogenous metabolic activation.

Interpretation of results: negative.

Executive summary:

Naphtha (petroleum), light alkylate (EC 265-068-8) was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay in the presence and absence of Aroclor induced rat liver S-9. Six mutagenesis assays were conducted. Three of the assays had technical difficulties and the results have not been presented. In the remaining three assays, for the S-9 activated cultures in particular, great difficulty was experienced in producing a smooth dose-response curve for toxicity. However, none of the cultures that were cloned, whether treated in the presence or absence of S-9, exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. Both non-activated and S-9 activated cultures were generated in the 10% to 50% Total Growth range.

The Total Growth of the cultures ranged from 18% to l43%.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

OECD 475 (on supporting substance with limited range, C7-C10): negative

Link to relevant study records

Reference

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: corn oil

Frequency of treatment:

single treatment

Post exposure period:

6, 24 and 48 hours

Remarks:

Doses / Concentrations:
0, 0.3, 1.0, 3.0 g/kg
Basis: nominal conc.

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes

Positive control(s):

- Positive control group: 500 µg/kg of triethylenemelamine (TEM), group exposed for 24 hours.
- Negative control group: corn oil (5 ml/kg body weight).

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Immediately after sacrifice bone marrow was obtained from the femurs of the animals. The marrow was washed and the cells were fixed before being spread on slides (at least 3 from each animal) for examination. Slides were scored for chromosomal aberrations. Where possible, a minimum of 50 metaphase cells from each animal were examined and scored for chromatid and chromosome gaps and breaks, fragments, structural rearrangements and ploidy (1-3). A mitotic index (= No. of cells in mitosis/500 counted X 100) was calculated and recorded.

Evaluation criteria:

The data were evaluated according to the following criteria:
For the test to be considered to be valid, the % of cells in the negative control group demonstrating aberrations of any type, other than gaps, must not exceed 4%. The % of cells with aberrations in the positive control group must be statistically increased (p=0.05) relative to the vehicle control using Chi-square statistics.
The test material is considered positive when the % of cells with aberrations in any treatment group is significantly increased (p = 0.05) relative to the vehicle control using Chi-square analysis and the number of aberrations per cell is also significantly increasd (p =0.05) relative to the vehicle control using t-test statistics.

Statistics:

see "Evaluation criteria"

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the cytogenetics assay, 5 of 18 males and 4 of 18 females receiving 3 g/kg Light Alkylate Naphtha died within 3 days. At this dose level, there was a weight loss of 10% and 9% in males and females respectively within 48 hours of administration. Other signs of toxicity included piloerection, crusty eyes and noses and excess lacrimation. No sex-related differences were noted in the study and therefore the data for males and females were combined for the cytogenetics evaluation.

Conclusions:

Interpretation of results (migrated information): negative
Naphtha (petroleum), light alkylate (EC 265-068-8) did not induce bone marrow chromosomal aberrations in male or female Sprague-Dawley rats.

Executive summary:

Naphtha (petroleum), light alkylate (EC 265-068-8) was tested in a Sprague Dawley rat chromosome aberration assay [15/sex/group] at doses of 0.3, 1.0, and 3.0 g/kg in corn oil, administered intraperitoneally in a single dose. Two to four hours prior to sacrifice the rats were given a single intraperitoneal dose of colchicine (1 mg/kg). Animals [5/sex/group/time] were sacrificed at 6, 24 and 48 hrs post dose. A group of 5 animals of each sex to be used as positive controls was dosed with triethylenemelamine (TEM) at a level of 0.5 mg/kg and these animals were killed at 24 hours postdose. Deaths occurred in both male [5/18] and females [4/18] in the highest dose group and a 9-10% body weight loss was observed in surviving rats of both sexes. Other signs of toxicity included piloerection, crusty eyes and noses and excess lacrimation. Bone marrow was harvested from the femurs of treated rats, processed and stained for cytogenetic examination [a minimum of 50 metaphase spreads per animal]. No chromosome aberrations, rearrangements, or cell cycle disruption were observed in any dose group.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Gene mutation in bacteria

According to the OECD guideline 471, the test material 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' was not mutagenic in Ames test with and without metabolic activation (tested up to 500 µg/plate in Salmonella typhimurium TA 100 without S9, up to 1500 μg/plate in TA100 with S9, up to 5000 µg/plate in TA1535, TA1537, TA 98 and in E.Coli wp2uvr A). No statistically significant increase in the numbers of revertant colonies was recorded for none of the bacterial strains with any dose of the substance, neither with nor without metabolic activation.

Gene mutation in mammalian cells

There is no in vitro data of the substance ‘Naphtha (Fischer-Tropsch), light, C4-C10 branched and linear’ available. However, a study was performed to assess the mutagenic toxicity in mammalian cells in vitro of the closely related substance ‘Naphtha (petroleum), light alkylate’ (covering the carbon numbers in the range C7-C10), whereby the used method was similar to OECD 490. "Naphtha (petroleum), light alkylate" did not induce mutations in the L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay, neither in the presence (concentration of test compound: 0.005 µl/ml - 0.04 µl/ml) nor in the absence of exogenous metabolic activation (concentration of test compound: 0.002 µl/ml - 0.75 µl/ml).

Cytogenicity in mammalian cells

In a GLP conform assay according to the OECD guideline 473 and draft proposal OECD guideline 487, human lymphocytes were tested for the detection of structural and numerical chromosomal aberrations in doses up to 1250 µg/mL of GTL Naphtha with and without metabolic activation in the first experiment and up to 117.2 mg/mL without and up to 1250 mg/mL with metabolic activation in the second experiment (Shell, 2006b). The method used is described in in the OECD guideline 487 in vitro micronucleus test but has been modified to more closely resemble the OECD guideline 473 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 toxic only in the 20-hour exposure group without metabolic activation, 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 or was limited by test material toxicity. The test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

In vivo

There are no in vivo data available for the substance ‘Naphtha (Fischer-Tropsch), light, C4-C10 branched and linear’. However, a study was performed to assess the genetic toxicity in vivo of the closely related substance ‘Naphtha (petroleum), light alkylate’ (covering the carbon numbers in the range C7-C10), whereby the used method was similar to OECD 475.

Accordingly, Light alkylate naphtha was tested in a Sprague Dawley rat chromosome aberration assay [15/sex/group] at doses of 0.3, 1.0, and 3.0 g/kg in corn oil, administered intraperitoneally in a single dose. Two to four hours prior to sacrifice the rats were given a single intraperitoneal dose of colchicine (1 mg/kg). Animals [5/sex/group/time] were sacrificed at 6, 24 and 48 hours post dose. A group of 5 animals of each sex to be used as positive controls was dosed with triethylenemelamine (TEM) at a level of 0.5 mg/kg and these animals were killed at 24 hours postdose. Deaths occurred in both male [5/18] and females [4/18] in the highest dose group and a 9-10% body weight loss was observed in surviving rats of both sexes. Other signs of toxicity included piloerection, crusty eyes and noses and excess lacrimation. Bone marrow was harvested from the femurs of treated rats, processed and stained for cytogenetic examination [a minimum of 50 metaphase spreads per animal]. No chromosome aberrations, rearrangements, or cell cycle disruption were observed in any dose group.

Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro and in vivo studies were negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo data the substance 'Naphtha (Fischer-Tropsch), light, C4-C10 branched and linear' is not genotoxic and does not require classification according to Regulation 1272/2008/EC.