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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-07-21 till 2005-11-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP. Some minor deviations from study plan due to updating of methods, as explained in the report. These deviations have no detrimental impact on the outcome of this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Some minor deviations from study plan were due to updating of methods and are explained in the report. These deviations have no detrimental impact on the outcome of this study.
GLP compliance:
yes (incl. certificate)
Remarks:
The study was performed in compliance with: ”Chemikaliengesetz“ (Chemicals Act) of the Federal Republic of Germany, ”Anhang 1“ (Annex 1), dated July 25, 1994 (”BGBl. I 1994“, pp. 1703), last revision dated June 27, 2002. ”OECD Principles of GLP
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MeNigu
- Physical state: solid, white
- Molecular weight: 118.1 g/mol
- Analytical purity: 99.7%
- Lot/batch No.: 9077154
- Expiration date of the lot/batch: June 1, 2007
- Stability under test conditions: not indicated by the sponsor
- Storage condition of test material: at room temperature, protected from light and moisture

Method

Target gene:
no data
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal essential medium with Hanks salts (MEM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
150, 300, 450, 600, 1200 mg/l
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; the final concentration of DMSO in the culture medium was 0.5 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative nontoxicity to the cell cultures.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in culture medium with 0.5 % [v/v] DMSO)

DURATION
- Preincubation period: 24 hours
- Exposure duration: without metabolic activation 4 and 20 hours, respectively; with metabolic activation 4 hours
- Expression time (cells in growth medium): without metabolic activation 24 hours; with metabolic activation 24 hours and 48 hours, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hours, respectively (depending on expression time)

SPINDLE INHIBITOR (cytogenetic assays): Colcemidband CPA, respectively
STAIN (for cytogenetic assays): the cells were stained with May Grünwald and Giemsa


NUMBER OF REPLICATIONS: For the micronucleus analysis the cultures of two flasks of each treatment group were seeded and treated in parallel


NUMBER OF CELLS EVALUATED: At least 2000 cells were scored per test group (except in Experiment I, in the absence of S9 mix, for the positive
control, where only 1000 cells were scored due to strong cytotoxicity)


DETERMINATION OF CYTOTOXICITY
- Method: For the assessment of cytotoxicity a XTT test was carried out in parallel to the micronucleus assay. The cells were seeded in the microtitre plates with approx. 20,000 cells per well for the 24 hours preparation interval (with and without S9 mix) and with approx. 10,000 cells per well for the48 hours preparation interval (with S9 mix). After treatment and preparation intervals identical to those of the micronucleus assay, 50 μL of the XTT labelling mixture was added to each well. This mixture consists of the XTT labelling reagent (5 mL) and an electron coupling reagent (0.1 mL). The cells were incubated for approx. 2.5 hours and subsequently transferred to a microplate equipped with a 450 nm filter to read the absorbance. To describe a toxic effect the relative cell count and the XTT activity of the test groups were determined as reduction of cells (in %) as compared to the respective solvent control.
Evaluation criteria:
A test item can be classified as mutagenic if:
− the number of micronucleated cells is not in the range of our historical control data (0.0 - 2.0 % micronucleated cells), and
− either a concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.

A test item can be classified as non-mutagenic if:
− the number of micronucleated cells in all evaluated test groups is in the range of our historical control data (0.0 - 2.0 % micronucleated cells).
and/or
− no concentration-related increase in the number of micronucleated cells is observed.
Statistics:
Statistical significance can be confirmed by means of the Chi square test.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant effects
- Effects of osmolality: no relevant effects
- Evaporation from medium: not applicable
- Water solubility: < 15 g/kg at 20 °C
- Precipitation: no precipitation observed


RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration applied (1200 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item following
the current OECD Guideline 473. In this study, in the absence and the presence of S9 mix, no cytotoxicity indicated by reduced cell numbers and/or XTT activities of below 40 % of control were observed after treatment with the test item.


COMPARISON WITH HISTORICAL CONTROL DATA:
Historical control data are given in the annex of the study report.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Summary of results of the micronucleus test with MeNigu (data in % micronucleated cells)
 Test item concentration in µg/ml  Exposure period 4 hrs without S9 mix (Exp. I) Preparation interval 24 hrs  Exposure period 20 hrs without S9 mix (Exp. II) Preparation interval 24 hrs  Exposure period 4 hrs with S9 mix (Exp. I) Preparation interval 24 hrs  Exposure period 4 hrs with S9 mix (Exp. II) Preparation interval 48 hrs
negative control  1.65  0.55 1.0  0.95 
solvent control   1.15 1.40   1.3  1.0
 positive control  69.20 *  41.40 *  n.d.  32.05 *
 positive control  n.d.  n.d. 7.2 *    n.d.
 150  n.d.   n.d.  n.d.  n.d. 
 300  n.d.   n.d.  n.d.   n.d.
 450  n.d.   n.d.  n.d.  n.d. 
 600  1.75  1.78  0.85  1.20
 900  1.65  0.90  1.0  1.10
 1200  1.40  1.53  1.5  1.30
* number of micronucleated cells statistically significant higher than corresponding control values n.d. not determined

Applicant's summary and conclusion

Conclusions:
The test item MeNigu did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation. Therefore, MeNigu is considered to be non-mutagenic in this in vitro test system when tested up to the highest required test item concentration.
Executive summary:

The test item MeNigu, dissolved in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro. Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation (S9 mix). In Experiment II the exposure period was 20 hrs without and 4 hours with metabolic activation. Per culture at least 1000 cells were scored for micronuclei. The highest applied test item concentration, 1.2 mg/mL, i.e.approx. 10 mM, was chosen with regard to the molecular weight of the test item following the current OECD Guideline 473.

In this study, in the absence and the presence of S9 mix, no cytotoxicity indicated by reduced cell numbers and/or XTT activities of below 40 % of control were observed after treatment with the test item. In the presence as well as in the absence of metabolic activation, no statistically significant or biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item with respect to the evaluation criteria mentioned in the current guidelines for in vitro genotoxicity studies. Appropriate mutagens were used as positive controls.

In conclusion, it can be stated that under the experimental conditions reported, the test item MeNigu did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation. Therefore, MeNigu is considered to be non-mutagenic in this in vitro test system when tested up to the highest required test item concentration.