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Diss Factsheets

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:
experimental study
Adequacy of study:
key study
Study period:
19 May 88 to 06 June 88
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
7,7-dimethyl-3-oxa-6-azaoctan-1-ol
EC Number:
400-390-6
EC Name:
7,7-dimethyl-3-oxa-6-azaoctan-1-ol
Cas Number:
87787-67-5
Molecular formula:
Hill formula: C8 H19 N O2 CAS formula: C8 H19 N O2
IUPAC Name:
2-[2-(tert-butylamino)ethoxy]ethan-1-ol
Details on test material:
- Name of test material (as cited in study report): MRD-88-192
- Physical state: Clear, colourless liquid
- Analytical purity: 100%
- Storage condition of test material: Room temperature

Method

Target gene:
Thymidine kinase (TK +/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Culture preparation: L5178Y TK +/- Mouse Lymphoma Cells were treated with methotrexate, thymidine, glycine and hypoxanthine prior to freezing to eliminate possible spontaneous TK -/- mutants. As needed, cells were then removed from liquid nitrogen storage, cultured and maintained in logarithmic phase for five days before use in the assay system.

Culture medium - F10p
Cloning medium - F20p
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Toxicity assay: Doses ranged from 1 to 1000 μg/ml.
Mutagenesis assay: Based on the results of the toxicity assay, six doses were used in the mutagenesis assay; 500, 700, 900, 1100, 1300 and 1500 μg/ml, but only five doses were plated. Because all doses had greater than 10% relative suspension growth, the lowest dose was eliminated; and 700, 900, 1100, 1300 and 1500 μg/ml were treated with and without activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle:Considered under the conditions of the assay to be stable for the duration of the assay.

The test material and positive controls were diluted and dosed prior to administration.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hour and 20 minute exposure
- Expression time (cells in growth medium): 2-day expression period


SELECTION AGENT (mutation assays): TFT = Trifluorothymidine Deoxyriboside


NUMBER OF REPLICATIONS: 3 plates per dose group.


NUMBER OF CELLS EVALUATED:
Approximately 9XE5 cells were plated in 30ml F20p with 2.5 μg/ml TFT. Concurrently, approximately 180 cells were plated in 30 ml F20p without a selective agent, to determine viable colony counts (VC).


DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growths, relative cloning growths, relative total growths, mutant frequencies amd induced mutant frequencies were calculated.

Plates were labelled with study number, compound identification number, the dose administered, VC or TFT, +S9 or -S9. Colonies were enumerated after eleven days incubation at 36-38 deg C with 4.5-5% CO2. Plate counts were made with a Biotran III colony counter and recorded.


Evaluation criteria:
A test material is considered positive if it yields a 2-fold increase over the spontaneous mutant frequency of vehicle controls at one or more dose levels, if a dose related increase in response is observed and if the relative total growth is greater than 10%. Should the relative total growth, (survival) be less than 10% and a two-fold increase in mutant frequency observed, the test material will be considered negative.
Statistics:
The following equations were used in the data analysis of the forward mutation assay:
Relative Suspension Growth, % =
Total suspension growth of test culture / mean total suspension growth of vehicle controls x 100%

Relative cloning growth, % =
Mean number of VC Colonies on “X” treated plates / Average of mean of VC colonies on vehicle control plates x 100%

Relative total growth, % =
(Relative suspension growth, %) (Relative cloning growth, %) / 100%

Mutant Frequency (mutants / 10E4) =
Mean number of TFT colonies for “X” / Mean number of VC colonies for “X” x 2

Induced Mutant Frequency (IMF) = Mutant frequency of treated cultures - mean mutant frequency of vehicle control cultures

VC = Viable colonies
"X" = Dose being evaluated

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
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:
Dose selection for the repeat mutation assay* was based on a toxicity (dose determination) assay. The doses selected were 700, 900, 1100, 1300 and 1500 μg/ml. These were tested with and without metabolic activation.

*The initial mutation assay was repeated because plate count values were out of range.

Please see attached background material for Table 1 - Summary of mouse lymphoma mutagenicity data, and Table 2 - Mouse growth results with and without metabloic activation.

A mutant frequency greater than 2(X) the mean control mutant frequency was not observed at any dose. The positive (DMBA and EMS) and negative (Controls 1 and 2, + and - S9) controls responded in a manner consistant with data from previos analysis.
Remarks on result:
other: strain/cell type: L5178Y cells
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

A mutant frequency that was greater than two times the mean vehicle control frequency was not observed at any dose level, nor was there any evidence of a dose related response.
For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.
Executive summary:

The mutagenic activity of the test substance was tested in the L5178Y mouse lymphoma assay. The positive controls were dimethylbenzanthracene (DMBA) and ethylmethanesulphonate (EMS). The substance was tested at 700, 900, 1100, 1300, 1500 μg/ml

with and without metabolic activation. Following a three hour and 20 minute exposure period, test and control cultures were allowed a two day expression period. Cultures were cloned in selective medium, then incubated for eleven days. Surviving and mutant colonies were then counted and the mutant frequencies calculated.

There was no evidence of a dose related increase in mutagenic frequency, and the mutant frequencies were not significantly different from control at any dose. For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.

The positive (DMBA, EMS) and negative (DMSO) controls responded in a manner consistant with data from previous studies.