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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:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted according to an appropriate guideline. No evidence of whether GLP was adhered to in the report.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Flux Oil (MCS 2170/Therminol 59)
Identification: Lot NBP 293350
EHL Code T850028
Stated Purity: Distilled
Stated Stability: Flux Oil was stated to be stable at room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO subclone KlBH4 originally obtained from Dr. A. W. Hsie of Oak Ridge National Laboratory, was used. The cells were routinely maintained in the laboratory as exponetially growing monolayer cultures in Ham's F12 medium (K. C. Biological) supplemented with heatinactivated 10% newborn calf serum (K. C. Biological) in an incubator-controlled environment with a 95% humidified atmosphere of 5% ±1% CO2 and 95% air at 37.5oC ±2oC.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate ( S 9 ) , lot no. 03830, commercially purchased from Litton Bionetics, was used as an exogenous activation system.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene (B(a)P) and ethyl methane sulfonate (EMS)
Evaluation criteria:
Positive: Reproducible dose-dependent mutagenicity with a slope statistically different (p 5 0.05) fromzero and with at least one point statistically higher (p 0.05) and > 2x of the negative control.

Negative: Reproducible negative mutagenicity tested to
cytotoxicity of approximately 10% relative survival or
limit of solubility, or, for nontoxic, soluble
chemicals, to 1 mg/rnl. Chemicals yielding a small,
non-dose related, positive response in Exp. 2 but
negative reponse in Exp. 3 will be considered as
non-mutagenic.

Inconclusive. Dramatically different response between
Exp. 2 and Exp. 3 (e.g., positive in Exp. 2 and
negative in Exp. 3) will not allow a definite
conclusion on the mutagenicity of the test substance.
On a case-by-case basis, repeat of the study may be
performed.

Exceptions: For highly cytotoxic or insoluble
chemicals, where only limited concentrations can be
tested, and only weak but statistically significant
mutagenicity is observed only at the highest possible
dose, scientific judgment will be made by the study
director to modify the experimental protocol to
determine if a dose-response relationship exists. The
mutagenicity of the test chemicals will be considered
inconclusive until dose-response relationship is
demonstrated.
Statistics:
Mutagenicity data were analyzed according to the statistical method of Snee and Irr (1981) designed specifically for the CHO/HGPRT mutation assay. Mutant frequency values were transformed according to the equation Y = (X + 1)0.15, where Y = transformed mutant frequency and X = observed mutant frequency. Student's t-test was then used to compare treatment data to solvent control data. The Snee and Irr analysis also allowed the determination of dose-response relationship as linear, quadratic, or higher-order. A computer program obtained from Dr. J. Irr (DuPont)d incorporated into the Monsanto computer by Allan Dickinson (Monsanto) was used.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
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
Remarks on result:
other: Flux Oil was concluded not to be mutagenic in CHO cells under the experimental conditions employed.

Any other information on results incl. tables

Initial cytotoxicity determination of Flux Oil in CHO cells at different S9 concentrations showed that the cytotoxicity of Flux Oil decreased with increasing S9 concentration. The concentrations of test material were 0, 0.3, 1.0, 3, 10, 33, 100, 333 and 1000 ug/ml. To confirm this observation, two additional range finding experiments were performed with the test material using narrow concentration ranges based on the treatment levels where toxicity was first observed. In the absence of S9, Flux Oil was found to be significantly cytotoxic (>50% cell killing) at 10 ug/ml. In the presence of 1 and 2% S9, Flux Oil was observed to be toxic at levels of 18 and 33 ug/ml, respectively. Some variation occurred in the cytotoxicity tests using 5% S9 when a range of treatment levels (47-100 ug/ml) yielded significant cell killing while higher levels of Flux Oil did not. Because of these variations, higher levels of Flux Oil were tested in the experiments conducted to determine the potential mutagenicity of the test material. No significant cytoxicity was observed when levels up to 1000 ug/ml were tested in the presence of 10% S9.

An initial experiment to determine the potential mutagenicity of the test material was conducted using a range of S9 concentrations. In this experiment Flux Oil was cytotoxic to the CHO cells at levels of 6 ug/ml and greater in the absence of exogenous metabolic activation. In the presence of S9 activation the cytotoxicity of the test sample decreased with increasing S9 concentrations.

A significant increase in cytotoxicity was demonstrated for treatment levels of 15 and 24 ug/ml in the presence of 1 and 2% S9 concentrations, respectively. No significant cytotoxicity was observed when Flux Oil was tested at maximum levels of 100 and 1000 ug/ml using 5 and 10% S9, respectively. A significant negative linear dose-response relationship (p<0.05) was observed in the treatments using 2% S9. The treatment doses of 24 and 48 ug/ml yielded a statistically significant (p<0.05) lower mutant frequency than the solvent control in the experiment with 2% S9. No statistically significant increases in mutant frequency were observed in any of the Flux Oil treated cultures.

The non mutagenicity of Flux Oil was confirmed by a subsequent experiment. In this experiment Flux Oil was tested at 1, 3, 6, 9, and 12 ug/ml in the absence of exogenous metabolic activation and at 10, 50, 100, 150, and 200 ug/ml in the presence of 5% S9. In the absence of S9 activation Flux Oil was observed to be cytotoxic at levels of 6 ug/ml and greater. In the presence of 5% S9, the test material was found to be significantly cytotoxic only at the highest level tested (200 ug/ml). No statistically significant increases in mutant frequency were observed in this experiment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

Flux Oil was tested in CHO cells at different S9 concentrations up to dose of 1000 ug/ml. No statistically significant mutagenicity was observed in two separate experiments. Flux Oil is, therefore, concluded not to be mutagenic in CHO cells under the experimental conditions employed.
Executive summary:

The mutagenic potential of Flux Oil was tested in cultured Chinese hamster ovary (CHO) cells. Mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing of Flux Oil was performed initially using a range of Aroclor 1254-induced rat liver homogenate (S9) concentrations (0-10%) followed by a confirmatory experiment at 0 and 5% S9. Flux Oil was shown to be cytotoxic at dose levels of 10, 18, and 33 ug/ml in the absence of S9 and in the presence of 1%, and 2% S9, respectively. The cytotoxicity was decreased significantly by increasing concentrations of exogenous Aroclor 1254 induced rat liver homogenate (S9) in the test system. Flux Oil was tested up to a dose of 1000 ug/ml with and without 5% metabolic activation. No test chemical- related mutagenicity was observed with and without metabolic activation. Flux Oil was therefore concluded not to be a mutagen in CHO cells under the experimental conditions of this assay.