Distillates (petroleum), hydrotreated middle

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: Study conducted to recognised method in GLP compliant laboratory

Justification for type of information:

The standard OECD 471 test is not suitable to test petroleum UVCBs, because it has a tendency to produce false negatives for these substances. Therefore, the petroleum industry has developed a Modified Ames assay, optimized to accurately identify positive results for this endpoint. This deviation from the prescribed testing procedure requires some further explanation which is given in the attached document. The document gives a brief history of the development of the Modified Ames test and outlines Concawe’s proposed work (as part of a wider testing strategy, see Annex 13) to further support the use of this test for PS.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Modified Ames Test according to ASTM E 1687

Principles of method if other than guideline:

14 Gas oils were examined for mutagenic activity in one histidine dependent auxotroph of Salmonella typhimurium, strain TA98, using a modification of the pour-plate assay designed to detect mutagenicity mediated by polynuclear aromatic compounds derived from petroleum, based upon the principles of the ASTM Standard Test Method E 1687.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

64742-46-7

Cas Number:

64742-46-7

IUPAC Name:

64742-46-7

Constituent 2

Reference substance name:

hydrotreated middle distillate

IUPAC Name:

hydrotreated middle distillate

Test material form:

other: low viscosity liquid hydrocarbon

Details on test material:

Sample name: Con#7
CAS No. 64742-46-7
Hydrotreated middle distillate

Sample name: Con#9
CAS No. 64742-80-9
Hydrodesulfurised middle distillate

Method

Target gene:

His- (reverse mutation to histidine-independence)

Species / strain

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

S. typhimurium TA98 hisD3052 rfa uvrB pKM101

Metabolic activation:

with

Metabolic activation system:

Aroclor 1254-induced S9

Test concentrations with justification for top dose:

Test substance
0, 12, 24, 36, 48, 60 microlitres per plate

Positive control
0, 3, 6, 9, 12, 15 microlitres per plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

concurrent DMSO extract of the Reference oil No. 1, a straight-run naphthenic vacuum distillate of known mutagenic activity

Details on test system and experimental conditions:

Preparation of test substance extract
Extracts of the test substance in dimethyl sulphoxide (DMSO) were prepared as follows:
Three separate aliquots (1 mL) of the test substance, warmed to approximately 45°C, were mixed thoroughly with 5 mL of DMSO. The mixtures were vortexed intermittently (every five minutes) for a total of thirty minutes. The mixtures were then centrifuged at 200 g for ten minutes, and the extract (lower phase) was removed. Only one extract of Reference oil No. 1 was prepared. All extracts were prepared on the day of use, and stored in the dark until required.
Dosing solutions were prepared for each test substance extract and for the reference oil extract by dilution with DMSO (the reference oil extract was diluted 1:3 with DMSO before preparation of the dosing solutions).

Mutation test procedure
The following sequence of steps was used in the performance of this test:
The S9 mix was prepared and placed on ice (for not longer than 2 hours before use).
“Top agar", consisting of 0.4% bacteriological grade agar and 0.5% NaCl in purified water (prepared by reverse osmosis) was melted and placed in a 45°C water-bath.
Dosing solutions for the test substance and Reference oil No. 1 were prepared as described above. Two (for each of the test extracts) or three (for the reference extract) sterile glass test tubes were dosed with 60 micro-L of each of these solutions and were allowed to stand for at least 30 minutes.
To each of the dosing tubes were added in order 0.5 mL of S9 mix and 0.1 mL of bacterial suspension, taking care to introduce both the S9 mix and the bacteria into the bottom of the tube. The tube was gently swirled after each addition. When each set of tubes (one test article including the solvent control) was completed, it was transferred to the gyratory incubator at 150 rpm and incubated at 37°C for 30 minutes.
The tubes were removed from the gyratory incubator. To each tube, beginning with the solvent controls and proceeding to higher doses, 2.0 mL of the top-agar, cooled to ca 40°C and supplemented with 10 mL of 0.5 mM histidine/0.5 mM biotin solution per 100 mL, was added. Immediately after addition, the mixture was vortex-mixed and poured onto previously prepared Petri dishes containing 25 mL minimal agar. Care was taken to obtain an evenly distributed and level top-agar layer. When the top-agar had hardened, the plates were inverted and incubated at 37°C for 48 hours. The plates were then removed from the incubator and the number of colonies determined by either automated or manual counting. Each Petri dish was individually labelled with a unique code recorded in the study file, identifying the contents of the dish.

Evaluation criteria:

For a test to be considered valid, the mean revertant colony counts obtained for Reference Oil No. 1 (diluted 1:3) must reach, in a dose-responsive manner, at least a two-fold increase over the mean solvent control count, and no more than three doses should produce mean revertant counts more than 15% below the following representative values:

Dose (µL/plate) 0 3 6 9 12 15

Mean revertants 46 54 69 73 81 93


The mean revertant count for the solvent controls should be in the range 30 - 60. Excursions from this range were considered acceptable only if there was no significant change in the slopes of the curves. The curve should be linear over at least four doses.

The Mutagenicity Index (MI), defined as the slope of the curve (revertants per µL DMSO extract), was calculated for all test samples.

Thresholds for interpretation of MI values defined in ASTM E 1687 are as follows:
Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.
Test substances with MI values >1 but <2 may or may not be non-carcinogenic in a mouse skin-painting bio-assay.
Test substances with MI values >2 are considered to have a high probability of being carcinogenic in a mouse skin-painting bio-assay.

A lower threshold value of 0.4 has been selected as the cut-off for Residual Aromatic Extracts (s), based on the results of skin-painting studies (Blackburn et al, 1996): s with a MI >0.4 demonstrated carcinogenic potential upon dermal application to mouse skin with chronic exposure, whereas s with a MI <0.4 did not demonstrate a carcinogenic potential.

Statistics:

The mean number of revertant colonies and standard deviations were calculated for all groups.
All valid data were plotted and analysed using a linear regression analysis programme.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

MI = 0.01

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Remarks:

not applicable, modified Ames protocol

Test results 3

Species / strain:

S. typhimurium TA 1537

Remarks:

not applicable, modified Ames protocol

Test results 4

Species / strain:

S. typhimurium TA 100

Remarks:

not applicable, modified Ames protocol

Test results 5

Species / strain:

S. typhimurium TA 102

Remarks:

not applicable, modified Ames protocol

Additional information on results:

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix and test substance extracts.
The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.
The mean revertant colony counts obtained for Reference Oil No. 1 (diluted 1:3) reached, in a dose-responsive manner, at least a two-fold increase over the mean solvent control count, and mean revertant counts were within the acceptable range.

It was, therefore, confirmed that the tests were valid.

Remarks on result:

other: conducted according to modified Ames protocol

Any other information on results incl. tables

Reference Oil No. 1
Dose (µL)	Counts			Mean	SD
0	45	43	44	44.0	1.0
3	52	50	50	50.7	1.2
6	66	65	68	66.3	1.5
9	71	71	74	72.0	1.7
12	80	77	78	78.3	1.5
15	86	90	91	89.0	2.6
Test sample Con#7
Dose (µL)	Counts A		Counts B		Counts C		Mean	SD
0	46	44	41	42	45	45	43.8	1.9
12	42	41	44	47	46	46	44.3	2.4
24	41	39	44	46	47	44	43.5	3.0
36	44	41	46	43	43	43	43.3	1.6
48	46	41	44	40	47	44	43.7	2.7
60	41	47	47	46	48	43	45.3	2.7

 

Con#7 MI value: 0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

The test substance had a Mutagenicity Index (MI) of 0.01

Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.

Executive summary:

The test substance had a Mutagenicity Index (MI) of 0.01

Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.