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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:
weight of evidence
Study period:
Oct 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,7-bis(diethylamino)phenoxazin-5-ium nitrate
EC Number:
277-539-5
EC Name:
3,7-bis(diethylamino)phenoxazin-5-ium nitrate
Cas Number:
73570-52-2
Molecular formula:
C20H26N3O.NO3
IUPAC Name:
3,7-bis(diethylamino)phenoxazin-5-ium nitrate
Test material form:
solid: particulate/powder

Method

Target gene:
Hypoxanthine-guanine phosphoribosyltransferase (HPRT1)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:MEM (minimal essential medium) with Hank's salts and 25 mM Hepes-buffer
- Properly maintained: ye
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat)
Test concentrations with justification for top dose:
Test groups with and without metabolic activation: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, 25, 50* µg/mL
* because of high toxicity in the main experiment, no mutant selection was performed

Control groups
negative controls:
a: untreated control
b: cultures treated with the solvent

positive controls:
a: without metabolic activation:EMS (Ethyl methane sulfonate)
b: with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)
Vehicle / solvent:
No vehicle, substance was directly dissolved into test system (MEM)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO for positive controls
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
In preliminary toxicity experiments approximately 1000 cells were seeded in each well of a microtiter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. After 4 to 5 days, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 1000 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of 4 or 5 days.

Two independent mutation tests were performed.

Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25% (v/v) trypsin ready for use (mfr. Gibco). A single cell suspension was prepared.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency (see above).

The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 1000 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6E5 - E6 cells in 182 cm² flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.

Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.
Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.
Day 5 or 6: Subculturing of b) in 182 cm² flasks
Day 9: Subculturing of b) in five 75 cm² flasks with culture medium containing 6-thioguanine: Mutant selection (about 300 000 cells/flask);
subculturing of b) in two 25 cm² flasks for plating efficiency (about 400 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.
All incubations were carried out at approx. 37°C and 4% CO2 , Staining was performed with approx. 10% (v/v) methylene blue in approx. 0.01% (w/v) KOH solution. Only colonies with more than 50 cells were counted.
Rationale for test conditions:
Based on results from preliminary study for solubility and toxicity
Evaluation criteria:
Criteria for a valid assay
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- the plating efficacy for the solvent control was greater than 50%

Criteria for a positive response
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
- survival of the responding dose group is at least 30%.

However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 25 µg/mL onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No increase of mutant colony numbers in two independent experiments.
Positive controls: valid
Cytotoxicity: yes, from 25 µg/mL in both with and without S9 mix

Applicant's summary and conclusion

Conclusions:
The test item did not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described (two independent experiments). The test item is therefore considered to be non-mutagenic in the HPRT assay with mammalian cells. The test item was shown to cause cytotoxic effects from 25 µg/mL onwards.