1H-1,4,7-Triazonine, octahydro-1,4,7-trimethyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 May 2008 to 23 September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in-vitro mammalian gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octahydro-1,4,7-trimethyl-1H-1,4,7-triazonine dihydrochlorid

Molecular formula:

C9H21N3, 2 HCl

IUPAC Name:

Octahydro-1,4,7-trimethyl-1H-1,4,7-triazonine dihydrochlorid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SAJ 07-065
- Expiration date of the lot/batch: 23 October 2009
- Purity test date:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at 2-8 °C
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: water >1 day at room temperature, > 2 days in the refrigerator, >3 days in the freezer
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: -

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Techni-cal University; 64287 Darmstadt, Germany
- Doubling time: 12-16h
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 15 mL MEM, 4.5% CO2
- 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:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I: 156.3, 312.5, 625.0, 1250.0, 2500.0
Exepriment II: 312.5, 625.0, 1250.0, 2500.0

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.5×10E6 (single culture) and 5×10E2 cells (in duplicate)

DURATION
- Preincubation period:
- Exposure duration: 4h (Experiment I), 24h (Experiment II)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: -

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen-trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corre-sponding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

4h treatment

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH was adjusted to 7.2
- Effects of osmolality: none
- Evaporation from medium: -
- Water solubility: -
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.

RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiment test item concentrations between 19.6 and 2500 μg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Following 4 hours treatment no toxic effects occurred up to the maximum concentration with and without metabolic activation. Following 24 hours treatment relevant toxic effect was observed at 1250 μg/mL and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (10E6 cells): EMS 4h (mean 163.2, SD 90.3, range 58.3-721.7), DMBA (mean 813.2, SD 428.2, range 94.9-2873.9), EMS 24h (mean 432.3, SD 343.6, range 61.8-1528.2)
- Negative (solvent/vehicle) historical control data: water 4h -S9 mix (mean 9.6, SD 5.7, range 1.7-31.1), water +S9 mix (mean 9.4, SD 5.1, range 1.1-29.1), water 24h -S9 mix (mean 10.9, SD 6.9, range 1.0-31.8)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effect as indicated by a relative cloning efficiency I of less than 50 % in both parallel cultures occurred up to the maximum concentration with and without meta-bolic activation following 4 h of treatment. After continuous treatment for 24 h without metabolic activation a minor cytotoxic effect was noted in the second culture at the maxi-mum concentration of the test item.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

In a mammalian cell gene mutation assay (HPRT) according to OECD testing guideline 476, V79 cells of the Chinese hamster cultured in vitro were exposed to TACN-Dichlorid at concentrations between 156.3 and 2500 µg/mL in the presence and absence of mammalian metabolic activation.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. A second experiment was performed without metabolic activation and a treatment period of 24 hours.

TACN-Dichlorid was tested up to cytotoxic or limit concentrations. The positive controls induced the appropriate response.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in both experiments. The threshold of three times the mutation frequency of the correspond-ing solvent control was not reached at any test point.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, TACN-Dichlorid is considered to be non-mutagenic in this HPRT assay.

This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.