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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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was conducted prior to the current OECD 476 guideline. The study design does not include a continuous exposure.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
The study design does not include a continuous exposure.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4',4''-(ethan-1,1,1-triyl)triphenol
EC Number:
405-800-7
EC Name:
4,4',4''-(ethan-1,1,1-triyl)triphenol
Cas Number:
27955-94-8
Molecular formula:
C20H18O3
IUPAC Name:
4-[1,1-bis(4-hydroxyphenyl)ethyl]phenol
Details on test material:
- Purity: >99+%

Method

Target gene:
thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RMPI 1640, supplemented with 0.1% Synperonic F68, 0.011% sodium pyruvate, 2 mM L-glutamine, 50 µg/mL gentamicin and buffered with 2 mg/mL sodium bicarbonate, is referred to as R0p. A variation buffered with HEPES called R0 (HEPES) was also used. A third media used was R0p supplemented with 10% HiDHS and referred to as R10p. R10p from which growing L5178Y cells had been removed was used as conditioned medium. R0 (HEPES) containing 5% HiDHS, designated R5 (HEPES), was used as the treatment medium. R0p in which the amount of Synperonic F68 had been reduced to 0.02% and supplemented with 30% HiDHS, referred to as R30p, formed the basis of the cloning medium. This was semi-solidified by the addition of Noble agar to a final concentration of approximately 0.4%. Selective medium consisted of cloning medium containing 4 µg/mL TFT.

- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced Sprague-Dawley rat liver homogenate (S-9)
Test concentrations with justification for top dose:
With and without activation, Test 1: 0, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, and 25 µg/m
With and without activation, Test 2: 0, 5, 10, 15, 20, 30, 40, 50, 60, and 80 µg/m
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: The maximum solubility of the test substance was 393.8 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
200 µL added to each culture
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methane sulphonate was used without activation, and 20-methylcholanthrene was used with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours after the 3 hour treatment period

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The criteria for a positive response were:
At least a 2-fold increase in mutant frequency in treated cultures relative to the control
Demonstration of a statistically significant increase in mutant frequency following treatment with the test substance
Evidence of a dose relationship over at least 2 dose levels, in any increase in mutant frequency
Demonstration of reproducibility in any increase in mutant frequency
The observed increases in mutant frequency must lie outside the upper limit of the historical control range, 150 mutants per 10E6 survivors.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al. (1989)

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the range-finding study, treatment with 1-200 µg/mL in the absence and presence of S-9 mix resulted in relative growth in suspension of 70-2% and 89-2% respectively compared to the solvent controls. Concentrations used in the main test were based upon this data.

COMPARISON WITH HISTORICAL CONTROL DATA: There were no increases in mutant frequency outside the upper limit of the historical control range, 150 mutants per 10E6 survivors.

Applicant's summary and conclusion

Conclusions:
The test substance did not demonstrate mutagenic potential in this in vitro gene mutation assay.
Executive summary:

The test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in a subline of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). Two independent tests in the absence of exogenous metabolic activation (S-9 mix) and two independent tests in the presence of S-9 mix were carried out. Toxicity was observed after treatment with the test substance in all the tests both in the absence and the presence of S-9 mix. No biologically significant increases in mutant frequency, according to the criteria used for a positive response, were observed after treatment with the test substance either in the absence or the presence of S-9 mix. Decreases in mutant colony size in comparison with the controls were not observed in any of the tests. It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.