Shale oils

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:

9th May - 23rd August 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Shale oils

EC Number:

269-646-0

EC Name:

Shale oils

Cas Number:

68308-34-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Shale oil

Details on test material:

Identity: Shale Oil
Chemical Name: Shale Oil
Batch No.: 1
Aggregate state at
room temperature: Liquid
Colour: Brown
Purity: At least 99 %
Stability in solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: March 04, 2006

Method

Target gene:

Histidine is the target gene for S. typhimurium strains.
Tryptophan is the target gene for E. coli strains

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: see materials and methods section

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: trp-; uvrA-:

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 1; 3; 10, 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used for test substance: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

- Vehicle(s)/solvent(s) used for positive control substances sodium azide and methylmethanesulfonate : deionised water
- Vehicle(s)/solvent(s) used for positive control substances 4-nitro-o-phenylene-diamine and 2-aminoanthracene : DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

T1537 and TA 98 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1 and pre-incubation for experiment 2

DURATION
- Preincubation period: 4 hours
- Preperation: In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
- Exposure duration: 2 hours

NUMBER OF REPLICATIONS: 3 replicates for each strain dose level and controls.

NUMBER OF CELLS EVALUATED: NDA

DETERMINATION OF CYTOTOXICITY
- Method: reductive in revertant colonies

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

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:

cytotoxicity

Remarks:

See table 2 for more information

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:

cytotoxicity

Remarks:

See table 2 for more information

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed in the two highest concentrations with all tester strains in Experiment II

DISCUSSION OF RESULTS

Reduced background growth was observed with and without metabolic activation in both independent experiments (cf. tables of results). Distinct toxic effects, evident as a reduction in the number of revertants (below the indicatation factor of 0.5), were observed as presented in Table 2 (below).

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Shale Oil at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

The laboratory´s historical control range was slightly exceeded in the solvent control of strain WP2 uvra with metabolic activation in experiment II. This minor deviation was judged to be based on biologically irrelevant fluctuation in the number of colonies and has no impact on the outcome of the study.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 2: Cytotoxicity Observed in Tester Strains

Strain	Experiment 1		Experiment 2
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1537	/	5000	5000	5000
TA 98	333 - 5000	2500, 5000	100 - 5000	1000 - 5000
TA 1535	1000	2500, 5000	333 - 5000	2500, 5000
TA 100	1000 - 5000	1000 - 5000	333 - 5000	1000 - 5000
Wp2 uvrA	5000	2500, 5000	5000	5000

/ no toxic effects observed

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Shale Oil is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Shale Oil to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The study was conducted according to OECD Guideline 471 and to GLP standard.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 1; 3; 10, 33; 100; 333; 1000; 2500; and 5000 μg/plate

Distinct toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment with Shale Oil at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.