Alkanes, C14-17-branched and linear

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June 1985 to 15 july 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Ames test (5 Salmonella strains), GLP. Substance analytical certificate available provided by the manufacturer. Substance identification: commercial name 98.8% purity

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

according to Ames test

Principles of method if other than guideline:

Guideline principles

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, C14-C17, n-alkanes, <2% aromatics

EC Number:

917-828-1

Cas Number:

185857-35-6

Molecular formula:

none available - not a single isomer - see remarks

IUPAC Name:

Hydrocarbons, C14-C17, n-alkanes, <2% aromatics

Constituent 2

Reference substance name:

Petrepar-147

IUPAC Name:

Petrepar-147

Details on test material:

- Name of test material (as cited in study report): Petrepar -147
- Substance type: petroleum product, UVCB
- Physical state: colourless liquid
- Analytical purity: 100% Commercial product
- Storage condition of test material: room temperature

Method

Target gene:

Reverse gene mutation assay

Species / strain

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:

other: S. typhimurium TA 1538 (see below Table 7.6.1/1)

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared in laboratory from liver of CD rat (Sprague-Dawley derived from Charles River Ltd, UK) induced by Aroclor 1254 and stored at -80 °C as aliquots.

Test concentrations with justification for top dose:

5, 50, 500, 5000 µg/plate in dose range-finding test (see below Table 7.6.1/2)
50, 50, 500, 1500, 5000 µg/plate in the main mutation tests

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

yes

Remarks:

Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of test substance, the S9 mix and the vehicle

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

yes

Remarks:

Sterile 0.1 M sodium phosphate buffer (pH 7.4)

Positive controls:

yes

Positive control substance:

other: See below Table 7.6.1/3

Remarks:

See freetext "Any other information on materials and methods"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) Two independent experiments (range-finding test as first and main test as second test) in agar by direct plate incorporation for both experiments with and without S9 mix.
Pre-incubation test substance activation was not performed in presence of S9-mix.

DURATION
- Preincubation period: no
- Exposure duration: 72 h

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: 3 measurements/plate) with a Biotran Automatic colony counter.


DETERMINATION OF CYTOTOXICITY
- Method: revertant colony counts during the dose-range finding assay


OTHER: scoring (3 measurements/plate) with a Domino automated counter

Evaluation criteria:

The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the negative (solvent) and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.

Statistics:

A compound is deemed to provide evidence of mutagenic potential if:
- a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and
- the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with Petrepar 147 at any dose level, either in the presence or absence of metabolic activation (S9).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration (µg/plate)	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	12	0.6	12	3.8	9	1.5	18	3.0	70	4.2
0**	9	0	10	1.2	7	1.0	21	7.0	66	3.5
50	9	2.0	12	3.6	9	1.2	17	1.0	72	6.1
150	8	3.1	7	1.5	9	1.2	15	4.2	69	4.9
500	8	2.0	11	3.2	5	0	15	3.5	72	3.8
1500	10	3.8	10	3.5	9	2.1	17	3.8	63	3.1
5000	9	2.1	11	3.5	12	1.0	18	3.5	63	5.0
Positive control***	65	5.5	1750	89.1	54	3.8	76	6.7	236	21.2

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration (µg/plate)	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	11	1.5	20	5.1	11	4.0	21	1.2	78	13
0**	9	2.3	21	2.1	9	1.5	14	1.5	69	4.0
50	7	0.6	11	1.0	9	4.2	16	3.6	72	5.1
150	8	2.0	10	2.1	9	2.3	13	1.5	69	6.0
500	6	2.0	12	3.0	9	3.1	14	3.5	71	4.7
1500	11	3.5	11	3.2	12	3.1	13	3.1	69	4.6
5000	6	1.0	9	3.8	9	1.5	13	2.3	61	2.9
Positive control***	130	9.0	103	17.4	408	47.5	372	52.4	542	15.9

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration (µg/plate)	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	14	2.0	14	3.8	5	1.2	14	3.2	63	2.9
0**	7	2.5	17	0.6	7	3.5	17	1.7	67	7..0
50	7	2.3	17	4.0	6	1.5	17	1.0	70	6.1
150	9	1.2	18	1.2	11	4.0	17	1.5	67	3.8
500	6	1.0	15	1.5	7	0	14	3.2	71	3.5
1500	6	2.1	19	2.0	8	2.9	16	2.6	72	4.0
5000	7	1.5	17	4.0	11	5.0	13	2.6	68	9.3
Positive control***	10	9.3	-	-	56	8.2	49	18.2	228	34.6

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Table 7.6.1/7: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (Second test)

Test substance concentration (µg/plate)	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Solvent*	10	2.6	14	3.8	9	2.6	19	2.6	88	17.9
0**	9	2.1	17	0.6	12	2.6	18	3.6	101	11.0
50	9	1.2	17	4.0	12	3.6	18	2.1	81	13.0
150	9	1.0	18	1.2	11	1.5	16	3.2	76	11.3
500	9	1.7	15	1.5	10	4.5	19	2.0	79	6.1
1500	9	3.1	19	2.0	11	4.0	16	2.6	79	9.1
5000	10	2.5	17	4.0	11	2.6	15	2.0	80	16.9
Positive control***	84	5.9	41	9.0	46	12.3	142	46.0	232	7.5

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

Under the test conditions, Petrepar-147 did not demonstrate any in vitro mutagenic activity in the Salmonella test system up to 5000 µg/plate.

Executive summary:

The mutagenic potential of Petrepar 147 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 mutants were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 72 hours of incubation at 37°C, the revertant colonies were scored.

A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The test substance was then tested in another experiment performed in the same way as the range-finding test.

The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies.
The test substance was dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared.

No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test).
Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

Under the conditions of this study, Petrepar 147 did not demonstrate any in vitro mutagenic activity in this bacterial test system.