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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
Study period:
From 12 January 2018 to 21 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29th July, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-2,5-dimethoxyfuran
EC Number:
211-797-1
EC Name:
Tetrahydro-2,5-dimethoxyfuran
Cas Number:
696-59-3
Molecular formula:
C6H12O3
IUPAC Name:
tetrahydro-2,5-dimethoxyfuran
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Remarks:
male
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: ECACC (European Collection of Cells Cultures), Lot. No. 10H016

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in an incubator with a humidified atmosphere, set at 5 % CO2.
- Cell cycle length, doubling time or proliferation index : doubling time 12-14 h
- Modal number of chromosomes: diploid number, 2n=22
Cytokinesis block (if used):
Colchicine (0.2 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH
- concentration or volume of S9 mix and S9 in the final culture medium : 250 µL S9 mix in 5000 µL (S9 fraction in S9 mix: 3 mL in 10 mL S9 mix, i.e. 30%)
Test concentrations with justification for top dose:
Based on the results of the preliminary cytotoxicity assay the following concentrations were selected for the Chromosome Aberration Assay:
Experiment A with 3/20 h treatment/sampling time:
- without: 125, 250, 500 and 1000 μg/mL test item
- with S9 mix: 62.5, 125, 250 and 500 μg/mL test item
Experiment B with 20/20 h treatment/sampling time, without S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 20/28 h treatment/sampling time, without S9 mix: 125, 250, 500 and 750 μg/mL test item
Experiment B with 3/28 h treatment/sampling time, with S9 mix: 62.5, 125, 250 and 500 μg/mL test item
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dissolved in DME (Dulbecco’s Modified Eagle’s) medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DME medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 105 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
Experiment A: 3-hour treatment, harvest 20 hours from the beginning of treatment
Experiment B without S9 mix: 20-hour treatment, harvest 20 hours from the beginning of treatment
Experiment B with & without S9 mix: 20 (without S9 mix)- and 3-hour (with S9 mix) treatment, harvest 28 hours from the beginning of treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine (0.2 μg/mL, 2.5 hours prior to harvesting)
- Methods of slide preparation and staining technique used including the stain used: Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Number of cells spread and analysed per concentration: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide).
- Criteria for scoring chromosome aberrations: Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative increase in cell count (RICC)
Evaluation criteria:
A test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
A the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Statistics:
For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps).
The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment dose using the adequate regression analysis by Microsoft Excel software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment A (3h/20h treatment/sampling time)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment B (20h treatment/sampling time)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment B (20h/28h treatment/sampling time)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment B (3h/28h treatment/sampling time)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.
In both experiments, clear cytotoxicity of about 50% was observed after test item treatment in the absence and presence of metabolic activation.

In experiment A in the absence of metabolic activation, no relevant increases in cells carrying structural chromosomal aberrations were observed. In experiment A in the presence of metabolic activation the cells with structural chromosome aberrations without gap at concentrations of 250 and 500 μg/mL were above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups, but this finding was considered biologically relevant.

In Experiment B, the frequency of the cells with structural chromosome aberrations without gap at the dose of 500, 750 μg/mL and 250 were above the 95% control limits of the historical control data up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.
Further, a 3-hour treatment at the doses of 62.5, 125, 250 and 500 μg/mL in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused a dose associated and biologically relevant increases in the number of cells with structural chromosome aberrations. Statistically significant difference were observed at the doses of 250 and 500 μg/mL when compared to the concurrent solvent as well as the historical control groups.
No increase in the rate of polyploid and endoreduplicated metaphases were found after treatment with the different concentrations of 2,5-Dimethoxytetrahydrofuran.
pH and osmolality values of control and test item treatment solutions were measured. In Experiments A and B no significant differences between test item treatment and control groups were observed.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Any other information on results incl. tables

TABLE 1: Mean number of cells with structural chromosome aberration(s) - Experiment A

Concentration (μg/mL)

S9 mix

Treatmenttime

Harvestingtime

Meanaberrantcells/150cells

incl. gaps

excl. gaps

Negative(Solvent) control

-

3 h

20 h

7

4

2,5-Dimethoxytetrahydrofuran

125μg/mL

-

3 h

20 h

9

4

250μg/mL

-

3 h

20 h

9

3

500μg/mL

-

3 h

20 h

10

3

1000μg/mL

-

3 h

20 h

11

4

Pos. Control (Ethylmethanesulphonate)

-

3 h

20 h

39**

32**

Negative(Solvent) control

+

3 h

20 h

7

4

2,5-Dimethoxytetrahydrofuran

62.5μg/mL

+

3 h

20 h

9

5

125μg/mL

+

3 h

20 h

7

4

250μg/mL

+

3 h

20 h

13

6

500μg/mL

+

3 h

20 h

17*

7

Pos. Control (Cyclophosphamide)

+

3 h

20 h

44*

39**

Positive control (-S9): Ethylmethanesulphonate(1.0μL/mL)

Positive control (+S9): Cyclophosphamide (5.0μg/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

* = p < 0.05 to the concurrent negative control and to the historical control

 

TABLE 2: Mean number of cells with structural chromosome aberration(s) - Experiment B (without S9 mix)

Concentration (μg/mL)

S9 mix

Treatmenttime

Harvestingtime

Meanaberrantcells/150cells

incl. gaps

excl. gaps

Negative(Solvent) control

-

20 h

20 h

8

14

2,5-Dimethoxytetrahydrofuran

125μg/mL

-

20 h

20 h

9

4

250μg/mL

-

20 h

20 h

10

3

500μg/mL

-

     20 h

20 h

11

5

750μg/mL

-

20 h

20 h

13

5

Pos. Control (Ethylmethanesulphonate)

-

20 h

20 h

43**

39**

Negative(Solvent) control

-

20 h

28 h

8

4

2,5-Dimethoxytetrahydrofuran

125μg/mL

-

20 h

28 h

12

4

250μg/mL

-

20 h

28 h

11

5

500μg/mL

-

20 h

28 h

11

4

750μg/mL

-

20 h

28 h

12

4

Pos. Control (Ethylmethanesulphonate)

-

20 h

28 h

45**

39**

Positive control (-S9): Ethylmethanesulphonate(0.4μL/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

 

TABLE 3: Mean number of cells with structural chromosome aberration(s) - Experiment B (with S9 mix)

Concentration (μg/mL)

S9 mix

Treatmenttime

Harvestingtime

Meanaberrantcells/150cells

incl. gaps

excl. gaps

Negative(Solvent) control

-

3 h

28 h

9

4

2,5-Dimethoxytetrahydrofuran

62.5μg/mL

-

3 h

28 h

18*

7

125μg/mL

-

3 h

28 h

22*

9

250μg/mL

-

3 h

28 h

21*/**

13*

500μg/mL

-

3 h

28 h

35**

20**

Pos. Control (Cyclophosphamide)

-

3 h

28 h

44**

38**

Positive control (+S9): Cyclophosphamide (5.0μg/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

* = p < 0.05 to the concurrent negative control and to the historical control

*/** = p < 0.05 to the concurrent negative control / to the historical control

 

Applicant's summary and conclusion

Conclusions:
2,5-Dimethoxytetrahydrofuran, tested up to the cytotoxic concentrations, induced structural chromosome aberrations in Chinese Hamster lung cells after a 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment.
Executive summary:

2,5-Dimethoxytetrahydrofuran was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity in accordance with the current OECD Guideline 473:

-       Experiment A with 3/20 h treatment/sampling time:

o      without: 125, 250, 500 and 1000 μg/mL test item

o      with S9 mix: 62.5, 125, 250 and 500 μg/mL test item

-       Experiment B with 20/20 h treatment/sampling time, without S9 mix: 125, 250, 500 and 750 μg/mL test item

-       Experiment B with 20/28 h treatment/sampling time, without S9 mix: 125, 250, 500 and 750 μg/mL test item

-       Experiment B with 3/28 h treatment/sampling time, with S9 mix: 62.5, 125, 250 and 500 μg/mL test item

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts.

In experiment A in the absence of metabolic activation, no relevant increases in cells carrying structural chromosomal aberrations were observed. In experiment A in the presence of metabolic activation the frequency of the cells with structural chromosome aberrations without gap at at concentrations of 250 and 500 μg/mL were above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups, but this finding was considered biologically relevant.

In Experiment B, the frequency of the cells with structural chromosome aberrations without gap at the dose of 500, 750 μg/mL and 250 were above the 95% control limits of the historical control data up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.

Further, a 3-hour treatment at the doses of 62.5, 125, 250 and 500 μg/mL in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused a dose associated and biologically relevant increases in the number of cells with structural chromosome aberrations. Statistically significant difference were observed at the doses of 250 and 500 μg/mL when compared to the concurrent solvent as well as the historical control groups.

There were no increases in the rate of polyploidy and metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

2,5-Dimethoxytetrahydrofuran, tested up to the cytotoxic concentrations, with mammalian metabolic activation system, induced structural chromosome aberrations in Chinese Hamster lung cells.

Thus, the test item is considered clastogenic in this system.