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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1st April to 21st May 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study following GLP. This study is being used as read across from Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased CAS No. 122384-87-6/68784-26-9 and so the reliability is reduced.

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 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
122384-87-6/68784-26-9/122384-86-5/68784-25-8. Phenol, dodecyl-, sulfurized, carbonates, calcium salts, overbased.
Testing was performed on a commercial sample of this material. Typical purity of this material as distributed in commerce is 50% alkyl phenol sulfide and 50% highly refined lubricant base oil.

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Strain: 3.7.2C
Cells were cultured in Fischer's medium in a shaker incubator at 37°C in humidified 5% CO2 in air. Cultures were diluted daily to a cell density of approximately 3 x 10ˆ5 cells per ml. Each time a culture was used, it was checked for bacterial or fungal contamination.
Prior to use in the assay, L5178Y cells were treated with methotrexate to reduce the frequency of spontaneously occurring TK-/- cells.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor S-9 metabolic activation
Test concentrations with justification for top dose:
0, 75, 100, 150, 200, 250, and 275 µg/mL with S-9
0, 60, 70, 80, 90, 100, and 110 µg/mL without S-9
Vehicle / solvent:
5% Pluronic F-68 (surfactant, CAS # 9003-11-6) in water.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Three replicate plates were evaluated for each treatment.

DURATION
- Test Material Exposure: Based on the data derived from the toxicity test, the test material was prepared so that the highest concentration would yield a percent total growth of approximately 10%. The test material was solubilized and diluted to produce evenly spaced dose levels which would yield approximately 90% total growth at the lowest dose. The test material was added to cells, both with and without metabolic activation and incubated for approximately 4 hours. Cells were then washed and placed into suspension culture at a density of 0.3 x 10ˆ6 cells/mL.

- Expression Time: In order for induced mutations to be expressed, the cells must undergo several divisions. This period is designated as the expression time. After the initial exposure to the test material the cells were incubated for 2 days and adjusted to 0.3 x 10ˆ6 cells/mL at 24 hours.

- Cloning: At the end of the expression period, a portion of the cells were plated in
(a) restrictive medium containing trifluorothymidine (TFT) which allowed only the TK-/- cells to grow, and
(b) non-restrictive medium which indicated cell viability.
The plates were incubated at 37°C in humidified 5% CO2 in air for 10-12 days.

- Accumulation of Data: After the incubation period, both the mutagenicity (TFT restrictive medium) plates and the viability (non-restrictive medium) plates were scored for the total number of colonies per plate using an Artek 880 colony counter or by hand. The frequency of mutants induced by each test material dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. Induced mutagenic activity, if any, was quantified by comparing the mutant frequency of the treated plates to that of the control plates.

DETERMINATION OF CYTOTOXICITY
The test material was checked for toxicity with and without S-9 metabolic activation at dose levels of 10 ug/ml to 5000 µg/ml. Toxicity was observed at 100, 500, 1000, 2500 and 5000 ug/ml with and without S-9.

Evaluation criteria:
Criteria for an Acceptable Mouse Lymphoma Screen: All the following criteria should be met for the mutagenicity screen to be considered valid:
- The mutant frequency of the appropriate positive control (either with or without S-9 activation) should be at least twice that of the appropriate negative (solvent) control.
- The spontaneous mutant frequency of the negative (solvents control should be in the range of 0.2 to 2.0 per 10ˆ4 cells.
- The plating efficiency of the negative (solvent) control should be at or above 50%.
- The test material should be tested to the level of approximately 10% total growth, or the limits of solubility, or to a high dose of 10 mg/ml. Test materials may be tested as slurries.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>100 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results of the preliminary Toxicity Test (not shown) conducted on the test material indicated significant toxicity at 100 µg/ml for the cultures with and without S-9 metabolic activation. Based on the results of the Toxicity Test, the test material was tested in the Mutagenicity Screen at concentrations ranging from 10 µg/ml to 275 µg/ml with metabolic activation and from 30 µg/ml to 110 µg/ml without activation. After the two-day expression period, six cultures with metabolic activation and six cultures without activation were selected for cloning based on the degree of observed toxicity. The cultures were cloned at 75, 100, 150, 200, 250, and 275 µg/ml (with metabolic activation) and 60, 70, 80, 90, 100, and 110 µg/ml (without activation).

None of the cultures treated with test material with or without metabolic activation exhibited mutant frequencies significantly different from the average mutant frequencies of the corresponding negative, solvent controls. Percent total growth ranged from 32% to 94% (with metabolic activation) and 10% to 95% (without activation). The positive control (DMBA) responded satisfactorily.

Under the conditions tested the test material was not mutagenic to L5178Y TK+/- cells in this assay.

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 without S-9, total growth only 32%. Total cell growth at the high dose without S-9 was 10%. Other test validity criteria were satisfied. The highest mutation frequency over control among all test doses was 4% with S-9 and 51% without S-9. There was no indication of a dose-related increase of mutation frequency with S-9. There was a trend without S-9 to higher mutation rates with progressively lower total growth at doses of 80-110 µg/mL. No statistical analysis was performed on the study results, as they were not needed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions tested the test material was not mutagenic to L5178Y TK+/- cells in this assay.
Executive summary:

In a study conducted in line with OECD guideline 476 and under conditions of GLP the test material was tested in the L5178Y TK+/- Mutagenicity Screen with and without S-9 metabolic activation. The cultures with activation were tested at concentrations ranging from 75 µg/ml to 275 µg/ml; cultures without activation were tested at concentrations ranging from 60 µg/ml to 110 µg/ml.

The results indicated that the test material did not induce a significant increase in the mutant frequencies of cultures tested either with or without metabolic activation. Under the conditions tested the test material was not mutagenic in this screen.

There was no available data to fulfil this endpoint for the test material and so the study was read-across from a supporting substance (Phenol, tetrapropenyl-, sulfurized, carbonates, calcium salts, overbased CAS No. 122384-87-6/68784-26-9).