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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:
Aug 28, 1996 - Jan 2, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: OECD guideline (not specified in report)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sure Sol®-300 (provided by Koch Industries Inc., Wichita, Kansas, USA)
(= compound of diisopropyl-1,1'-biphenyl and tris(1-methylethyl)-1,1'-biphenyl, ~80% and ~20%, resp., acronym: di-IPB/tri-IPB)
- Substance type: organic
- Physical state: clear, colorless liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Lot/batch No.: no data
- Storage condition of test material: room temperature, protected from light, under nitrogen

Method

Target gene:
Thymidine kinase locus TK +/-

Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
heterozygous at the thymidine kinase (TK) locus; designated as clone 3.7.2C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 (prepared from male Sprague-Dawley rats)
Test concentrations with justification for top dose:
Preliminary dose range finding experiment: 500 µg/ml (highest concentration) followed by nine lower concentrations prepared in two-fold dilution steps with each vehicle (with and without metabolic activation).
Main experiment: see table below (14 concentrations between 2.5 and 80 µg/mL)
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol, acetone (concentration in medium: 1%)
- Justification for choice of solvent/vehicle:
Controls
Negative controls:
yes
Remarks:
without test item, with and without S9
Solvent controls:
yes
Remarks:
ethanol or acetone, with and without S9
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl methanesulfonate (MMS; 5.0 nl/ml and 10 nl/ml, without S9); methylcholanthrene (MCA; 2.0 µg/ml and 4.0 µg/ml, with S9)
Details on test system and conditions:
Independant repeat trials were performed with duplicate cultures using either ethanol or acetone as vehicle.
Cells from exponentially growing cultures were seeded in a series of tubes (approx. 6 x 10E6 cells per tube); after pelleting by centrifugation
resuspension in 10 ml of treatment medium, incubation at 37°C in an orbital shaker incubator.

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: no data
- Exposure duration: 4 h, 37°C
- Expression time (cells in growth medium): 48 h, 37°C
- Selection time (if incubation with a selection agent): 10 - 14 d

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT, 3 µg/ml) in cloning medium

NUMBER OF REPLICATIONS: 2x per dose level and assay, vehicle control: 3x per assay, positive control: 2x per assay

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:
Minimum criterion considered necessary for mutagenicity of any treatment:
- Mutant frequency equal or higher the 2-fold the concurrent background mutant frequency (= average mutant frequency of vehicle control)

Further criteria must be fullfilled:
- A dose-related or toxicity-related increase in mutant frequency for at least three dose steps
- Signifiant mutagenic activity should be confirmed in replicates of the same dose level
- A mutagenic dose-response in one assay should be confirmed in a second assay

A test article is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or, in the case of relative nontoxic materials, a range of applied concentrations extending to the maximum of 500 µg/ml

Assay acceptance criteria:
- Average absolute cloning efficiency of the vehicle controls should be between 60% and 130%.
- Minimum value for suspension growth of the average vehicle controls: 8.0-fold incrase in two days from the original cell number
- Mutation frequency of positive controls must be must be equal or higher than 200 x 10E-6

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Precipitation: White precipitate was observed in culture medium at concentrations between 35.0 µg/ml and 500 µg/ml

RANGE-FINDING/SCREENING STUDIES:
- Vehicle ethanol ( with and without metabolic activation): weakly cytotoxic to noncytotoxic in a dose range from 0.977 µg/ml to 31.3 µg/ml,
cytotoxic at 62.5 µg/ml, and lethal at higher concentrations.
- Vehicle acetone (without metabolic activation): moderately cytotoxic in a dose range from 0.977 µg/ml to 7.81 µg/ml.
Higher concentrations were highly cytotoxic or lethal.
- Vehicle acetone (with metabolic activation): weakly cytotoxic to noncytotoxic in a dose range from 0.977 µg/ml to 15.6 µg/ml, highly cytotoxic
at 31.3 µg/ml, and lethal at higher concentrations.

Trial 1 with acetone as vehicle (without metabolic activation) was terminated, because a good range of cytotoxicity was not obtained for analysis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item was negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation under the conditions of
testing.