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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Principles of method if other than guideline:
The procedure and experimental design are in accordance with the following guideline/ references:
- A proposal for a new OECD Guideline for the in vitro micronucleus test, 1998;
- Mut. Res., 439, 183 - 190, 1999;
- Mut. Res., 303, 163 - 169, 1993;
- Toxicol. in Vitro, 7, 185 - 193, 1993
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): PGMS
- Analytical purity: 92.3 %
- Lot/batch No.: Versuch 83
- Stability under test conditions: according to the analytical report, the test substance was stable over the study period
- Storage condition of test material: Room temperature

- Physical state/ appearance: solid / yellow
- Shape of particles: spherical
- Aspect ratio: not specified
- Particle size distribution: not specified
- Crystal structure: crystalline
- Surface area of particles: not specified
- Surface treatment: no

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (with Earle's salt)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S9 mix
Test concentrations with justification for top dose:
Mixed Population Method (MP):
- 24 hours exposure, 24 hours harvest time, without S-9 mix: 0, 6.25, 12.5, 25, 50, 100 µg/ml
- 4 hours exposure, 24 hours harvest time, with S-9 mix: 0, 31.25, 62.5, 125, 250, 500 µg/ml
Mitotic Shake off Method (MSO) (24 hours mitotic shake off):
- 24 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, without S-9 mix: 0, 6.25, 12.5, 25, 50, 100 µg/mI
- 4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0, 125, 250, 500, 1000, 1500 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

PROCEDURE:
- Mixed Population Method (MP): The experimental procedure was carried out based on the method of KALWEIT, et al. (Mut. Res., 439, 183-196, 1999).
- Mitotic Shake off Method (MSO): The experimental procedure was carried out based on the method of SEELBACH, A. et al. (Mut. Res., 303, 163 - 169, 1993; and Toxicol. in Vitro, 7, 185 - 193, 1993).
- 1000 cells were analyzed for micronuclei for each culture, i.e. 2000 cells for each test group.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei;
- The proportion of micronucleus-containing cells exceeded both the concurrent negative control range and the negative historical control range.

A test substance is generally considered nongenotoxic in this test system if:
- There was no significant increase in the number of micronucleus-containing cells at any dose above concurrent negative control frequencies and within the historical 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was observed macroscopically depending on the test conditions and on the time of observation from about 200 µg/mI culture onward.

RANGE-FINDING/SCREENING STUDIES:
The doses for the Mixed Population Method (24 hours sampling time) were determined from an appropriate range-finding cytotoxicity test with cultures exposed to a wide dose range of the test article, i.e. 1.0 µg/ml - 1500 µg/ml culture medium both without S-9 mix (continuous treatment of 24 hours) and after adding a metabolizing system (pulse treatment of 4 hours). In this pretest, various parameters were checked for all or at least some selected doses. As a rule, the highest dose 1 concentration for nontoxic test substances should not exceed a limit of 5 mg/ml or 10 mM, as recommended by the EEC, OECD and by an ICPEMC Task Group for in vitro studies using mammalian cells. Doses > 1500 µg/ml led to an inhomogeneous suspension which could not administered any longer. On the basis of the findings from the pretest, 100 µg/ml (0.136 mM) without S-9 mix and 1000 µg/ml (1.362 mM) with metabolic activation were selected as the top doses. This selection was based on the cell count, cell attachment, solubility of the test substance and quality of the cells.

RESULTS:
- The negative controls (vehicle controls) gave frequencies within the range expected for the V79 cell line.
- Both of the positive control chemicals for clastogenicity, i.e. ethyl methane sulphonate and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei both in the MP and MSO.
- Micronucleus Frequency: An increase in the number of micronucleated cells was not found either without S-9 mix or after the addition of a metabolizing system.
- Mitotic Index (MI): no suppression of the mitotic activity was observed under any of the experimental conditions.
- Proliferation Index (PI): a weak cytotoxic response was observed only without S-9 mix at 100 µg/ml.
- Cell Count: a dose-dependent growth inhibition was observed under all experimental conditions from about 100 µg/ml - 125 µg/ml onward.
- Micronucleus Frequency: An increase in the number of micronucleated cells was not observed either without S-9 mix or after the addition of a metabolizing system.

Any other information on results incl. tables

Table 1: The micronucleus frequency in MP method based on 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation


















































































































Hours



Test groups



S9 mix



Mean


(% absolute)



Exposure



Harvest



24



24



Vehicle control, DMSO



-



0.5



24



24



6.25 µg/ml



-



1.0



24



24



12.50 µg/ml



-



0.6



24



24



25.00 µg/ml



-



0.7



24



24



50.00 µg/ml



-



0.9



24



24



100.00 µg/ml



-



1.2



24



24



350.00 µg/ml



-



5.6



4



24



Vehicle control, DMSO



+



0.6



4



24



31.25 µg/ml



+



0.5



4



24



62.50 µg/ml



+



1.0



4



24



125.00 µg/ml



+



0.4



4



24



250.00 µg/ml



+



0.9



4



24



500.00 µg/ml



+



0.9



4



24



2.5 µg/ml CPP



+



18.9



 


Table 2: The micronucleus frequency in 24-h Mitotic Shake Off method based on 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation

































































































































Hours



Test groups



S9 mix



Mean


(% absolute)



Exposure



Mitotic Shake Off



Harvest



24



24



27



Vehicle control, DMSO



-



0.6



24



24



27



6.25 µg/ml



-



0.8



24



24



27



12.50 µg/ml



-



0.5



24



24



27



25.00 µg/ml



-



0.7



24



24



27



50.00 µg/ml



-



0.9



24



24



27



100.00 µg/ml



-



0.7



24



24



27



350.00 µg/ml



-



6.1



4



24



27



Vehicle control, DMSO



+



0.5



4



24



27



125.00 µg/ml



+



0.9



4



24



27



250.00 µg/ml



+



1.0



4



24



27



500.00 µg/ml



+



1.0



4



24



27



1000.00 µg/ml



+



1.3



4



24



27



1500.00 µg/ml



+



1.3



4



24



27



2.5 µg/ml CPP



+



15.4



 

Applicant's summary and conclusion

Conclusions:
On the basis of the results of the present study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S-9 mix or after adding a metabolizing system. These findings were confirmed in two modified versions of the in vitro micronucleus assay carried out independently of each other.
Thus, under the experimental conditions of this assay, the test substance is considered not to be a chromosome-damaging (clastogenic) agent, nor does it induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.
Executive summary:

The test substance was assessed in V79 cells in vitro for possible clastogenic or aneugenic activity leading to inducement of micronuclei both in the presence and in the absence of a metabolizing systemn.

According to an initial range-finding cytotoxicity test tor the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiment, the following doses were evaluated:

Mixed Population Method (MP):

24 hours exposure, 24 hours harvest time, without S-9 mix: 0; 6.25; 12.5; 25; 50; 100 µg/ml

4 hours exposure, 24 hours harvest time, with S-9 mix: 0; 31.25; 62.5; 125; 250; 500 µg/ml

Mitotic Shake Off Method (MSO) (24 hours mitotic shake Off):

For confirmation of the results of the 1st experiment (Mixed Population Method), the following doses were analyzed:

24 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, without S-9 mix: 0; 6.25; 12.5; 25; 50; 100 µg/ml

4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 125; 250; 500; 1,000; 1,500 µg/ml

Test substance precipitation was observed macroscopically depending on the test conditions and on the time of observation from about 200 µg/ml culture onward.

1,000 cells were analyzed for micronuclei for each culture, i.e. 2,000 cells for each test group.

The negative controls (vehicle controls) gave frequencies within the range expected for the V79 celI line.

Both of the positive control chemnicals for clastogenicity, i.e. ethyl methane sulphonate and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei both in the MP and MSO.

On the basis of the results of the present study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S-9 mix or after adding a metabolizing system. These findings were confirmed in two modified versions of the in vitro micronucleus assay carried out independently of each other.