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Toxicological information

Genetic toxicity: in vitro

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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:
5th to 15th April 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study following GLP.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name and of test material (as cited in study report): Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased
- CAS number of test material (as cited in study report): 68784-26-9
Testing was performed on a commercial sa4mple of this material. Typical purity of this material as distributed in commerce is 50% alkyl phenol
sulfide carbonate and 50% highly refined lubricant base oil.

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100 and TA 102 & E. coli WP2 uvrA
Details on mammalian cell type (if applicable):
Species and cell type: Male Sprague-Dawley rat, liver S-9 fractionQuantity: 25 µl/plate
Additional strain / cell type characteristics:
other: Histidine-deficient strains of Salmonella typhimurium and a tryptophan-deficient strain of Escherichia coli were grown.
Metabolic activation:
with and without
Metabolic activation system:
0.5 mL of 5% v/v S-9 mix per plate Induced or not induced: Aroclor 1254 induced, 500 mg/kg i.p.
Test concentrations with justification for top dose:
0, 0.033, 0.1, 0.33, 1.0, and 3.33 mg/plate, with and without S-9
Vehicle / solvent:
The vehicle used was 25% w/w Pluronic 127 (surfactant, CAS # 9003-11-6) in ethanol.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: See table in materials and methods section below.
Details on test system and experimental conditions:
METHOD OF APPLICATION: All media and solutions were prepared as described by Maron and Ames (1983). Plate incorporation test.
Three replicate plates were evaluated for each treatment.

DURATION
The bacterial tester strain culture was grown by inoculating nutrient broth with a tester strain and incubating with shaking at ~37°C to a density of 1-2 x 10ˆ9 cells/ml. Without metabolic activation, 100 µl of tester strain and 100 µl of solvent or test material were added to 2.5 ml of molten selective top agar at ~45°C. With metabolic activation, 100 µl of tester strain, 100 µl of solvent or test material, and 0.5 ml of S-9 mix were added to 2.0 ml of molten selective top agar at ~45°C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 hours at ~37°C. The number of revertant colonies was counted and the presence and condition of the bacterial lawn was noted. Solvent and positive controls were run concurrently.

Test material was not completely miscible with the top agar at >5 mg/plate. The test material mixture for the highest dose level (10 mg/plate) was too viscous to deliver accurately.
Testing at the highest pipettable dose level (5 mg/plate) with 25-400 µl S-9/plate on strains TA98 and TA100 indicated that increasing the concentrations of S-9 did not signficantly increase the induction of revertants; therefore, 25 µl S-9/plate was used for the mutagenicity assay.

DETERMINATION OF CYTOTOXICITY
- Method: The test materials were checked for toxicity on strain TA100 with and without S-9 mix at dose levels of 0.003 to 10 mg/plate. No toxicity was observed.
Evaluation criteria:
The following criteria were met before the results of the mutagenicity assay were judged as positive or negative:
(1) the high dose showed some toxicity, or was 10 mg/plate or the limit of solubility;
(2) the spontaneous controls were within the laboratory's acceptable range;
(3) the positive controls produced at least a three-fold increase in the number of revertants over the values for the respective negative controls.


Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 98, TA 100 and TA 102 & 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:
When tested at concentrations of 0.033 to 3.33 mg/plate the test material was not mutagenic to TA98, TA100, TA102, or E. Coli WP2 uvrA with or without metabolic activation provided by Aroclor-induced rat liver S-9. No cytotoxicity was observed at any concentration. The apparent increase in strain TA102 with S-9 metabolic activation is not considered biologically significant; the mean ± SEM for historical solvent control values is 325 ± 30.

The tester strains responded to the positive controls, known mutagens, as expected.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

First criterion for a valid test (as stated in the report) was not met. Dose of 5 mg/plate was the highest pipettable dose level and the solubility limit in top agar. Other test validity criteria were satisfied. No information was presented on condition of the background lawn, which would help assessment of cytotoxicity. Nevertheless, highest test dose of 3.33 mg/plate was close to the solubility limit and clearly not cytotoxic or mutagenic, based on revertants/plate data. The greatest mean number of revertants/plate over concurrent control among all test doses and all strains was 42% with S-9 and 19% without S-9. There was no indication of a dose-related decrease or increase of mean revertants/plate. Statistical results were not applicable.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions tested, the test material was not mutagenic to TA98, TA100, TA102, or E. coli WP2 uvrA.
Executive summary:

In a GLP compliant study conducted in line with the standard methodology published by Ames, the test material was tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, and TA102 and in the tryptophan-deficient strain of Escherichia coli WP2 uvrA at dose levels of 0.033 to 3.33 mg/plate with and without metabolic activation provided by Aroclor-induced rat liver S-9. The test material was suspended in 25% Pluronic 127 (w/w in ethanol). The suspension was miscible with the top agar. It was not cytotoxic at any concentration tested.

Under the conditions tested, the test material was not mutagenic to TA98, TA100, TA102, or E. coli WP2 uvrA.