[No public or meaningful name is available]

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:

weight of evidence

Study period:

March 14, 2012 - May 03, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Renewable hydrocarbons (diesel type fraction)

EC Number:

700-916-7

Molecular formula:

UVCB substance, not available. View remarks field.

IUPAC Name:

Renewable hydrocarbons (diesel type fraction)

Details on test material:

- Name of test material (as cited in study report): Diesel
- Substance type: UVCB substance
- Physical state: liquid
- Analytical purity: 100%; UVCB substance
- Lot/batch No.: 11-09149-001
- Expiration date of the lot/batch: 01 January 2013
- Storage condition of test material: room temparature in the dark

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

A preliminary range-finding assay
TA100 and WP2uvr with and without metabolic activation (micrograms/plate)
5000, 1500, 500, 150, 50, 0

Main assay 1 (micrograms/plate)
All strains (with and without S9-mix) :5, 15, 50, 150, 500, 1500, 5000

Main assay 2 (micrograms/plate)
All strains (with and without S9-mix) :5, 15, 50, 150, 500, 1500, 5000

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at 100 mg/ml.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial cultures and S-9 mix or phosphate buffer and vehicle or test item were incubated for 20 min at 37 deg. C. Two milliliters molten Top Agar, trace histidine or trypthophan, were added to the mixtures. The mixture was plated on Minimal Agar plates and incubated for 48 hours at 37 deg. c.

Evaluation criteria:

Following criteria are used for determining a positive result:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear negative or positive results, in some instances the data generated will prohibit making a definitive judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data is conducted as determined by UKEMS (Mahon et al., 1989, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.) Cambridge University Press Report, 26-65.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the highest test item concentrations tested (5000 micrograms/plate) without metabolic activation. No cytotoxicity was observed at any concentration tested with metabolic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the highest test item concentrations tested (5000 micrograms/plate) without metabolic activation. No cytotoxicity was observed at any concentration tested with metabolic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

No cytotoxicity was observed with or without metabolic activation up to the test item concentration of 5000 micrograms per plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the highest test item concentrations tested (5000 micrograms/plate) without metabolic activation. No cytotoxicity was observed at any concentration tested with metabolic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the highest test item concentrations tested (5000 micrograms/plate) without metabolic activation. No cytotoxicity was observed at any concentration tested with metabolic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: immiscible
- Precipitation: test item precipitate (oily in appearance) was noted at 5000 micrograms per plate.


RANGE-FINDING/SCREENING STUDIES: The test item was not toxic to TA100 and WP2uvr strains when tested up to 5000 micrograms per plate in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: See attached background material.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test item was tested for mutagenic potential using in vitro bacterial reverse mutation test. The test substance did not exert mutagenic activity both in the presence and the absence of metabolic activation.

Executive summary:

The test item was tested for the mutagenic potential using in vitro bacterial reverse mutation test (Ames test)(corresponding to the OECD No. 471). A preliminary range-finding assay was performed using four strains of Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and one strain of E. Coli (WPuvrA) up to a maximum dose of 5.0 mg/plate to determine the optimal non-toxic test dose. The test item was either tested up to the maximum recommended dose level of 5.0 mg/plate or the toxic limit depending on bacterial strain type and presence or absence of S9-mix.

Test item did not produce any significant increase in mutation frequency in any of these strains, with any dose of the test item, either in the absence or presence of metabolic activation. Based on the study results the test substance is considered to be non-mutagenic.

All the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 mix and the sensitivity of the bacterial strains.

The test result is used as a key value in the hazard assessment.