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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
Type of information:
experimental study
Adequacy of study:
key study
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Version 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Version 19 May 2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
Version Aug 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary
Specific details on test material used for the study:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Analytical purity: 95%
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- First experiment: 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 µg/mL (with and without S9 mix); only 0, 2.0, and 4.0 µg/mL test groups were evaluated for chromosomal aberrations.
- Second experiment: 0.5, 1.0, 2.0, 3.9, 7.8, 15.8, 31.3, 62.5, 125.0, 500.0, and 1000.0 µg/mL (with and without S9 mix); only 0, 1.0, 2.0 and 3.9 µg/mL test groups
were evaluated for chromosomal aberrations.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: in comparison to other commonly used vehicles (e.g. water, acetone etc.), DMSO was the most suitable one in the previous testing on formulability of the test substance and was therefore selected as vehicle. DMSO has been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available. However, due to the physico-chemical properties of the test substance, the highest achievable concentration in DMSO was 100 mg/mL. The final concentration of the vehicle DMSO in the culture medium was 1 % (v/v).
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
historical control data from the testing laboratory were available
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
CA tests are regulary conducted in the testing laboratory
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
historical control data from the testing laboratory were available
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
CA tests are regulary conducted in the testing laboratory
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; all cultures were prepared in duplicate

DURATION
- Exposure duration: 4 hours with or without S9 mix (main study); 4 and 18 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix (pretest).
- Expression time (cells in growth medium): 14 hours (sampling time was 18 hours [~1.5-fold cell cycle time])
- Fixation time (start of exposure up to fixation or harvest of cells): 20 - 36 hours; subsequently to colcemid treatment, the culture medium was completely removed. For hypotonic treatment, about 5 mL of a prewarmed 0.4 % (w/v) KCl solution (37 °C) was added for about 20 minutes. Subsequently, about 5 mL of fixative (methanol:glacial acetic acid, ratio 3:1; +4 °C) was added. After at least 15 minutes, the solution was removed and replaced by fresh fixative. After about another 10 minutes, the fixative was replaced again and kept at room temperature for at least 5 minutes. The slides were taken out of the Quadriperm chambers, briefly allowed to drip off and then rapidly passed through a Bunsen burner flame.

SPINDLE INHIBITOR (cytogenetic assays): 2 - 3 hours prior to cell harvest, 100 μL colcemid (stock: 10 μg/mL colcemid dissolved in PBS) was added to each chamber in order to arrest mitosis in the metaphase.

STAIN (for cytogenetic assays): after drying, the slides were stained with 7.5 % (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes and mounted in Corbit-Balsam.

NUMBER OF REPLICATIONS: triplicates

NUMBER OF CELLS EVALUATED: 100
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. In the case of clearly increased aberration rates, the number of metaphases to be analyzed for this test group can be reduced to at least 50 metaphases per culture. In addition, numerical chromosome aberrations were scored and recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth
A mitotic index based on 1000 cells/culture was determined for all evaluated test groups in all cytogenetic experiments. As additional parameter for cytotoxicity, the cell count was determined by means of test cultures prepared especially for this purpose in 25 cm² flasks for all test groups, except the positive controls. Growth inhibition was estimated by counting the number of cells in the test groups at the end of the culture period using a cell counter

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
* Polyploidy: changes in the number of chromosomes by whole chromosome set.
* Aneuploidy: metaphases with absent (hypoploid) or additional (hyperploid) chromosomes. Only hyperploid metaphases were recorded.
- Determination of endoreplication: yes
* Endopolyploidy: tetraploid metaphases with so-called diplo-chromosomes (products of endomitotic chromosome reduplication).

OTHER:
- Cell morphology: at the end of the treatment period, the test cultures of all test groups, except the positive controls, were checked microscopically (100x magnification) for cell morphology (which is an indication of an attachment of the cells to the slides).
- pH value: changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the vehicle control with and without S9 mix.
- Osmolarity: osmolarity was measured, at least for the top dose and for the vehicle control with and without S9 mix.
- Solubility: possible test substance precipitation was checked immediately after treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).
Evaluation criteria:
The V79 in vitro cytogenetic assay was considered valid if the following criteria are met: (1) the quality of the slides allowed the identification and evaluation of a sufficient number of analysable metaphases. (2) The numbers of cells with structural/numerical aberrations in the negative control was within the range of the historical negative control data. (3) The positive control substances both with and without S9 mix induced a distinct increase of structural chromosome aberrations.
The test substance is assessed as “positive” if the following criteria are met: (1) a statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps). (2) The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met: the number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
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 and osmolality: osmolarity and pH values were not influenced by test substance treatment.
- Precipitation: in the 1st experiment strong test substance precipitation was observed at least at the highest concentrations tested on the slides until staining of the cells. However, after clarification of the slides in xylene the test substance precipitation was lost. Therefore, there was no interference of the evaluation for cytogenetic damage in the highest applied concentrations. Finally, the dose selection of the 1st experiment did not fulfill the requirements of the current OECD Guideline 473. Thus, in the repeat experiment, designated 2nd Experiment, concentrations up to the border of formulability (1000 μg/mL) were applied.
- Other confounding effects: cell attachment was slightly reduced (i.e. few cells rounded) from about 62.5 μg/mL onward in the 2nd Experiment with or without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
On the basis of the chemical properties of the test substance the highest concentration that could be formulated in an appropriate vehicle (DMSO) and was applied in the pretest was 1000 μg/mL (approx. 2.6 mM). Test substance precipitation in the vehicle was observed from the lowest applied concentration (test group 1 μg/mL) onward. In culture medium test substance precipitation determined macroscopically occurred at 3.9 μg/mL and above at the end of treatment in all experimental parts. In the absence and the presence of S9 mix at 250 μg/mL and above, the cell attachment could not be assessed because test substance particles strongly covered the cell layer. Based on this observation it was expected that evaluation for cytogenetic damage in spread metaphase cells will be distinctly interfered at these concentrations. pH value and osmolarity were not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured in the pretest.
After 4 hours treatment in the absence of S9 mix, cytotoxicity indicated by reduced cell numbers of below 50 % of control was observed at 1000 μg/mL. In contrary, in the presence of S9 mix, no clear cytotoxicity was observed. In the pretest with 18 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 500 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The dose selection was made on the basis of the data and the observations from the pretest (e.g. cytotoxicity only at high concentrations showing strong test substance precipitation; expectation of interference between precipitation and evaluation for cytogenetic damage) and taking into account the current guidelines.
Remarks on result:
other:
Remarks:
a statistically significant increase in the number of chromosomally damaged cells was observed, but no relevant increase in the number of cells with changes in the number of chromosomes was demonstrated.

Any other information on results incl. tables

According to the results of the present in vitro cytogenetic study, the test substance led to a dose-dependent and biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps with metabolising system after a treatment time of 4 hours and a sampling time of 18 hours. The concentrations 2.0 μg/mL (5.5 % aberrant metaphase cells, exclusive gaps) and 3.9 μg/mL (6.8 % aberrant metaphase cells, exclusive gaps) were at the borderline or exceeded the aberration rates of the historical negative control data range (0 - 5.5 % aberrant metaphase cells, exclusive gaps). These observations were corroborated by scoring the respective concentrations (2.0 and 4.0 μg/mL) of the 1st experiment with S9 mix (7.5 and 6.0 % aberrant metaphase cells, exclusive gaps, respectively). The scoring of the positive control group in this experimental part was not necessary. Therefore, the observations in the presence of metabolic activation have to be regarded as biologically relevant for the in vitro cytogenetic study.

In the 2nd Experiment in the absence of S9 mix after a treatment time of 4 hours and a sampling time of 18 hours a slight dose-related increase of aberrant metaphase cells compared to the respective vehicle control was observed in the concentration range scored (1.0 to 3.9 μg/mL). However, the values were nearby the respective vehicle control value and were well within the historical negative control data range (0 - 5.5 % aberrant metaphase cells, exclusive gaps). Therefore, this observation has to be regarded as biologically irrelevant. The negative outcome of this experimental part was not corroborated by scoring the respective test groups due to the positive finding in the experimental part with S9 mix. In this study, no relevant increase in the number of cells containing numerical chromosomal aberrations was observed in the absence and the presence of metabolic activation. The structural chromosome aberration rates of the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study.

The increase in the frequencies of structural chromosome aberrations induced by the positive control substances and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

 

Table 1: First experiment; summary of the results (only the evaluated 3 dose groups in the presence of S9 mix are shown)

Schedule exposure/ preparation period

Test groups

Genotoxicity

Cytotoxicity*

Aberrant cells [%]

Polyploid cells [%]

Cell number [%]

Mitotic index [%]

incl. gaps#

excl. gaps#

with exchanges

4/18 hours

Vehicle control1

5.5

2.0

1.5

1.0

100.0

100.0

2.0 µg/ml

10.5

7.5S

4.0

2.4

101.7

114.2

4.0 µg/ml

8.5

6.0S

2.0

0.5

84.8

135.3

Precipitation was observed from 2.0 µg/ml onwards; *: relative values compared with the respective vehicle control; #: inclusive cells carrying exchanges;S: aberration frequency statistically significant higher than corresponding control values;1: DMSO 1 % (v/v)

 

Table 2: Second experiment; summary of the results (only the evaluated 4 dose groups in the presence and absence of S9 mix are shown)

Schedule exposure/ preparation period

Test groups

Genotoxicity

Cytotoxicity*

Aberrant cells [%]

Polyploid cells [%]

Cell number [%]

Mitotic index [%]

incl. gaps#

excl. gaps#

with exchanges

4/18 hours

With S9 mix

Vehicle control1

9.0

3.0

1.0

1.0

100.0

100.0

1.0 µg/ml

6.0

0.0

0.0

1.4

101.6

114.5

2.0 µg/ml

10.3

5.5

2.3

1.7

110.9

149.7

3.9 µg/ml

12.0

6.8

3.3

1.6

95.7

133.1

Positive control2

23.0S

18.0S

9.0S

0.0

n.t.

149.0

4/18 hours

Without S9 mix

Vehicle control1

8.0

3.5

1.5

2.0

100.0

100.0

1.0 µg/ml

7.5

3.0

0.5

0.5

83.2

135.8

2.0 µg/ml

8.0

4.0

2.5

0.5

86.0

101.7

3.9 µg/ml

7.8

4.3

1.5

1.2

80.8

142.5

Positive control2

13.0

12.0S

4.0

2.0

n.t.

135.0

Precipitation was observed from 2.0 µg/ml onwards; *: relative values compared with the respective vehicle control; #: inclusive cells carrying exchanges;S: aberration frequency statistically significant higher than corresponding control values;1: DMSO 1 % (v/v);2:500 μg/ml; n.t.: not tested

 

Applicant's summary and conclusion