Condensation products of oil shale alkylresorcinols and formaldehyde

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

Study period:

23 March 2004 to 09 April 2004

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:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

447-830-3

EC Name:

-

Molecular formula:

Not applicable to this UVCB substance.

IUPAC Name:

2-ethylphenol; 3,5-dimethylphenol; formaldehyde; phenol

Test material form:

other: solid resin

Details on test material:

- Date received: 30 January 2004
- Storage conditions: Room temperature in the dark under nitrogen

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

The strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Mutation test 1: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Mutation test 2: TA100, TA1535, TA1537 with & without S9: 15, 50, 150, 500, 1500, 5000 μg/plate. TA98, TA102 with & without S9: 50, 150, 500, 1500, 5000 μg/plate.

Vehicle / solvent:

Solvent: Dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

Remarks:

in absence of metabolic activiation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA), 1,8-Dihydroxyanthraquinone (DAN)

Remarks:

in presence of metabolic activiation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

PREPERATION OF TEST AND CONTROL MATERIALS
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials were as follows:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 μg/plate for TA100 and 5 μg/plate for TA1535
9-Aminoacridine (9AA): 80 μg/plate for TA1537
Mitomycin C (MMC): 0.5 μg/plate for TA102
4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate for TA98

In addition, 2-Aminoanthracene (2AA), Benzo(a)pyrene (BP) and 1,8-Dihydroxyanthraquinone (DAN), which are non-mutagenic in the absence of metabolising enzymes, were used in the S9 series of plates at the following concentrations:

2AA: 1 μg/plate for TA100
2AA: 2 μg/plate for TA1535 and TA1537
BP: 5 μg/plate for TA98
DAN: 10 μg/plate for TA102

MICROSOMAL ENZYME FRACTION
S9 was prepared in-house on 28 February 2004 from the livers of male Sprague-Dawley rats weighing approximately 250g. These had each orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at -196 ºC.

S9-MIX AND AGAR
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADPH 2.0 mL
0.1 M NADH 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 12.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin solution added to each 100 mL of top agar.

TEST PROCEDURE
PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel- Bonner Minimal agar (30 mL/plate). Ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MUTATION TEST 1
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

MUTATION TEST 2
The second experiment was performed using methodology as described for Mutation Test 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, based on results from Mutation Test 1.

Evaluation criteria:

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
• All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls
• The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
• All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per mL
• Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix

EVALUATION CRITERIA
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. This is considered to be a positive result.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material was toxic at and above 500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test material was toxic at and above 500 μg/plate to the strain of Salmonella used (TA100).
The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile.

MUTATION TESTS:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9, initially from 1500 μg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. An orange colour was noted from 500 μg/plate with an associated precipitate observed at 5000 μg/plate. Neither of these observations prevented the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.
All of 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

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

Executive summary:

The genetic toxicity of the test material was examined in an Ames bacterial reverse mutation assay using Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100, with and without metabolic activation.

The method was designed to meet the requirements of the OECD 471, EU Method B.13/14 and the USA, EPA (TSCA) OPPTS harmonised guidelines. The test was conducted to GLP standard, in a certified laboratory.

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 caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9, initially from 1500 μg/plate.

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