Phenol, paraalkylation products with C10-15 branched olefins ( C12 rich) derived from propene oligomerization, calcium salts, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalyc dewaxed, light or heavy paraffinic C15-C50

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:

18th September to 1st December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study following GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

UV sensitivity of cultures not verified in all experiments; this deviation from the guideline shown (by historical repeatability of results using the same frozen stock cultures) not to affect the validity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, paraalkylation products with C10-15 branched olefins ( C12 rich) derived from propene oligomerization, calcium salts, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalyc dewaxed, light or heavy paraffinic C15-C50

EC Number:

701-249-4

Molecular formula:

A molecular formula for this substance does not exist because it is a UVCB. The molecular formula for a theoretical representative structure is C36H58Ca2O4Sx where x = 1-3.

IUPAC Name:

Phenol, paraalkylation products with C10-15 branched olefins ( C12 rich) derived from propene oligomerization, calcium salts, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalyc dewaxed, light or heavy paraffinic C15-C50

Details on test material:

Phenol, dodecyl-, sulfurized, calcium salts
Testing was performed on a commercial sample of this material. Typical purity of this material as distributed in commerce is 60% alkyl phenol sulfide and 40% highly refined lubricant base oil.

Method

Target gene:

no data

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

100, 250, 500, 1000, 5000 and 10000 µg/plate, with and without S-9

Vehicle / solvent:

25% w/w Pluronic F127 (surfactant, CAS # 9003-11-6) in ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Vehicle was tested as negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table below.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Bottom agar (25 ml per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose.

DURATION
- Preincubation period:
Overnight cultures for use in all testing procedures, will be inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks will be placed in a shaker/incubator which will be programmed to begin operation (shaking, 125 ± 25 rpm; incubation, 37 ± 2°C) so that the overnight cultures are in log phase or late log phase when turbidity monitoring begins.
- Exposure duration:
In the plate incorporation methodology, the test article, the tester strain and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation at 37 ± 2 °C for 48 ± 8 hr, revertant colonies were counted. All doses of test article, vehicle controls, and positive controls were plated in triplicate.


DETERMINATION OF CYTOTOXICITY
- Method: The growth inhibitory effect (cytotoxicity) of the test article to the test system will be determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay.

Evaluation criteria:

The following criteria will be used to determine a valid assay:
1.Tester Strain Integrity: Salmonella typhimurium
a. uvrB Excision Repair Deletion: To demonstrate the presence of the ma B excision repair deletion, tester strain cultures must exhibit sensitivity to UV light.
b. rfa Wall Mutation: To demonstrate the presence of the rfa wall mutation, tester strain cultures must exhibit sensitivity to crystal violet.
c. pKM 101 Plasmid: To demonstrate the presence of the R-factor plasmid, pKM101, cultures of tester strains TA98 and TA100 must exhibit resistance to ampicillin.
d. Characteristic Number of Spontaneous Revertants: To demonstrate the requirement for histidine, the tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions.

2. Tester Strain Integrity: Escherichia coli
a. uvrA Excision Repair Deletion: To demonstrate the presence of the uvrA excision repair deletion, tester strains must exhibit sensitivity to UV light.
b. Characteristic Number of Spontaneous Revertants: To demonstrate the requirement for tryptophan, the tester strain culture must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. The acceptable range for the WP2uvrA mean vehicle control is 5 to 40 revertants per plate.
3. Tester Strain Culture Density: To demonstrate that appropriate numbers of bacteria are plated, the density of tester strain cultures must be greater than or equal to 0.5 x 10ˆ9 bacteria per ml and/or have reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 10ˆ9 bacteria per ml.

4. Positive Control Values
a. Positive Control Values in the Absence of S9 Mix
b. Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity)

5. Cytotoxicity
A minimum of three non-toxic dose levels will be required to evaluate assay data

Statistics:

no data

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Precipitation: Test article precipitate was observed on the plates at 5.00 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic concentration:
With metabolic activation: > 10,000 µg/plate
Without metabolic activation > 10,000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
Doses to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test article using tester strains TA 100 and WP2uvrA in both the presence and absence of a 10% S9 mix (one plate per dose). Ten doses of test article, from 5,000 to 6.67 µg per plate, were tested. These data were generated in Experiment 17865-Al. No cytotoxicity was observed up to 5,000 µg per plate in the dose rangefinding study with tester strains TA100 and WP2uvrA in either the presence or absence of S9 mix as evidenced by a normal background lawn and no reduction in the number of revertants per plate. Test article precipitate was observed on the plates at 33.3 µg per plate and above with both tester strains in both the presence and absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

All test validity criteria were satisfied. There was no evidence of cytotoxicity based on condition of the background lawn or revertants/plate data at doses up to 10,000 µg/plate. The greatest mean number of revertants/plate over concurrent controls among all test doses, all strains and for both assays was 100% (TA 1537, next highest 81%) with S-9 and 100% (TA 1535 and 1537, next highest 47%) without S-9. There was no indication of a dose-related decrease or increase of mean revertants/plate.

Applicant's summary and conclusion

Conclusions:

Not mutagenic with or without metabolic activation

Executive summary:

The mutagenic potential of the substance was assessed in an "Ames" test conducted under conditions of GLP and in accordance with OECD guideline 471. The results of the Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that, under the conditions of this study, in both an initial and a confirmatory assay, the test article did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor^TM-induced rat liver (S9). No cytotoxicity was observed up to 10,000 µg/plate with the Salmonella tester strains or with WP2uvrA in either the presence or absence of S9 mix. Test article precipitate was observed on the plates at 5.00 µg/plate. The test is therefore considered to have a negative result and the substance is considered non-mutagenic under the conditions of teh test in both the presence and absence of metabolic activation.