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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:
weight of evidence
Study period:
14 September 2009 to 01 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The cell line used was Chinese hamster ovary (CHO 10 B4) cells, obtained from the University of Leiden in 1987. The cell line is mycoplasma tested (in house) on a regular basis. The cells were grown as monolayers, and have a generation time of approximately 12 h. The modal chromosome number has been determined for these cells to be 21. The cells were incubated at 37°C.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Colcemid was added to all cultures at a final concentration of 0.1 μg/mL.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
The highest concentration tested was 10 mM (1041 µg/mL). This showed no change in osmolality or changes to the colour of the media. No observations of precipitation were made.
Vehicle / solvent:
Ham's F-10 medium
Controls
Untreated negative controls:
yes
Remarks:
Ham's F-10 medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Cells were trypsinised from stock flasks at passage numbers 15 (Test 1) and 12 (Test 2), and resuspended in fresh culture medium at densities of 0.1 x 10^6 or 0.05 x 10^6 cells/mL. These cells, in 5 mL volumes, were dispensed into 25 cm2 tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment respectively. Test cultures were established from the stock flask about 20 h before testing.

Tests were conducted both in the presence and absence of S9 mix. Treatments with test item or vehicle control substances were performed on duplicate cell cultures. Several concentrations of the positive controls were tested using single cultures.

Test 1 comprised treatments in the absence and presence of S-9 for 6 hours with recovery and harvest at 24 hours.
Test 2 comprised treatments in the presence of S-9 for 6 hours with recovery and harvest at 24 hours and treatments in the absence of S-9 for 22 hours with no recovery and harvest at 24 hours or included a recovery period with harvest at 48 hours.

Cultures to be treated in the presence of S9 mix were washed before treatment with serum free medium. Exposure medium was prepared, immediately before dosing, in sterile containers.

After treatment, cells were washed twice with serum free medium, then full growth medium added, for the recovery period and colcemid treatment. The volume of medium for the recovery period was 5 mL. S-9 was included at 10% v/v, where applicable.

Living cultures were examined for evidence of changes to cell morphology, once at the end of the treatment period and again before harvesting of cultures.

Colcemid was added to all cultures at a final concentration of 0.1 μg/mL. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase; the stage of cell division at which chromosomes can be examined using light microscopy. Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approximately 190 g), and treated with hypotonic solution (1% trisodium citrate) for 15 min at room temperature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 3:1). Two further changes (after sedimentation as before) of fixative were made.
Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity.
For both experiments, 3 slides per culture were made. All slides were marked with the study number and assigned a unique, coded number from a computer generated sequence. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with 5% Giemsa, then made permanent by mounting coverslips with DPX mountant.

Slides were examined for evidence of metaphase cells and signs of cellular necrosis.

Three concentration levels were selected for assessment of chromosomal aberrations.

From 2 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells per culture, were examined where possible. Slides were scored in order of coded number. A reduced number of metaphases were scored if a high proportion (≥40%) of metaphase cells were found to be damaged. A microscope was used for this assessment, the magnification used being x 1000 or x 1250, achieved with x 10 or x 12.5 eyepieces and x 100 objective. The number of chromosomes in each metaphase cell and all abnormalities, using the nomenclature of Gebhart (1970) was recorded. The types of structural and numerical aberrations recorded are listed in abbreviations page. The positions on the slides of any structurally aberrant cells were recorded using the Vernier scale on the microscope stage.

As cultures harvested at both culture times were negative with regards to structural aberrations, a further assessment of polyploidy was made. The Study Director selected the later harvest time as being more appropriate for this analysis. This decision was based on results from the previous assessments. For this assessment, approximately 300 metaphase cells were cursorily examined at a magnification of x 400 or x 500 and deemed to be either diploid, polyploid or endoreduplicated. In this assessment no metaphase cell in a field was rejected.
This assessment was considered more objective, avoiding scorer selection of either normal or polyploid cells. In addition, the larger sample size gave a more accurate frequency of such cells in the population.

From the cell counts, the number of cells recovered per culture, was calculated. This was then compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures to give a measure of toxicity.

From the results, 5 parameters were calculated, and judged as negative, suspicious or positive.
These parameters were:
1. Lesions per cell
2. Percentage of aberrant cells including cells with gaps only
3. Percentage of aberrant cells excluding cells with gaps only
4. Percentage of aneuploid cells
5 Percentage of polyploid cells (normal and endoreduplicated) from additional assessment of polyploidy
The third parameter is considered the most important in judging the true clastogenicity of a test item.

The results obtained were compared with the historical control data.
The experiments in this study were deemed to be valid because they fulfilled the following criteria:
There was no evidence of contamination
Cells in vehicle control cultures had normal growth
The results of vehicle and positive control cultures were typical
The test item had 3 acceptable dose levels for assessment
Evaluation criteria:
A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.

The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historica negative control data. The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% conf dence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.

A test was positive if the response in at least one acceptable dose level was significant by the criterion described above. A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of abe rant cells (between 95% and 99% confidence limits). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.
Statistics:
No statistical analysis was performed.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Results: Experiment 1 - 6 hour treatment in the presence of S-9, 24 hour harvest

Concentration (µg/mL)

Replicate

Index (% of mean vehicle control cell count)

N

Number of structural aberrations

Frequency of structural aberrations (%)

% of cells with

Ctg

Ctb

Ctf

Csg

Csb

Csf

Cse

Other

With gaps

Without gaps

AE

ER

PP

0 (vehicle control)

A

96

100

0

0

0

0

0

0

0

0

0

0

0

0

1

B

104

100

0

0

0

0

0

0

0

0

0

0

0

1

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0.5

0.5

260

A

102

100

0

0

0

0

0

0

0

0

0

0

0

1

0

B

96

100

0

0

0

0

0

0

0

0

0

0

0

1

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

1

0

521

A

91

100

0

0

0

0

0

0

0

0

0

0

0

1

0

B

95

100

0

0

0

0

0

0

0

0

0

0

0

2

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

1.5

0

1041

A

91

100

0

0

0

0

0

0

0

0

0

0

0

3

0

B

87

100

0

0

0

0

0

0

0

0

0

0

0

2

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

2.5

0

Cyclophosphamide

40 µg/mL

A

-

100

5

3

0

0

0

0

8

0

13

9

2

0

0

Cyclophosphamide

50 µg/mL

A

-

100

2

13

1

0

0

0

11

0

21

21

7

0

0

 

Results: Experiment 1 - 6 hour treatment in the absence of S-9, 24 hour harvest

Concentration (µg/mL)

Replicate

Index (% of mean vehicle control cell count)

N

Number of structural aberrations

Frequency of structural aberrations (%)

% of cells with

Ctg

Ctb

Ctf

Csg

Csb

Csf

Cse

Other

With gaps

Without gaps

AE

ER

PP

0 (vehicle control)

A

100

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

100

100

1

0

0

0

0

0

0

0

1

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0.5

0

0

0

0

260

A

97

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

97

100

0

0

0

0

0

0

0

0

0

0

1

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0.5

0

0

521

A

97

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

87

100

0

0

1

0

0

0

0

0

0

1

0

0

0

Total

-

200

0

0

1

0

0

0

0

0

0

0.5

0

0

0

1041

A

73

100

0

0

0

0

0

0

0

0

0

0

1

0

0

B

77

100

0

0

0

0

0

0

0

0

0

0

1

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

1

0

0

Methyl methane-sulphonate

30 µg/mL

A

-

100

2

4

0

0

0

0

2

0

8

6

0

0

1

Methyl methane-sulphonate

40 µg/mL

A

-

100

1

4

3

0

0

0

2

0

9

8

0

0

0

 

Results: Experiment 2 - 6 hour treatment in the presence of S-9, 24 hour harvest

Concentration (µg/mL)

Replicate

Index (% of mean vehicle control cell count)

N

Number of structural aberrations

Frequency of structural aberrations (%)

% of cells with

Ctg

Ctb

Ctf

Csg

Csb

Csf

Cse

Other

With gaps

Without gaps

AE

ER

PP

0 (vehicle control)

A

96

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

104

100

0

0

0

0

0

1

0

0

1

0

1

1

0

Total

-

200

0

0

0

0

0

1

0

0

0.5

0

0.5

0.5

0

260

A

93

100

0

0

0

0

0

0

0

0

0

0

0

1

0

B

100

100

0

0

1

0

0

0

0

0

1

1

1

1

0

Total

-

200

0

0

1

0

0

0

0

0

0.5

0.5

0.5

0

0

521

A

93

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

98

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

1041

A

102

100

0

0

0

0

0

0

0

0

0

0

2

0

1

B

81

100

0

0

0

0

0

0

0

0

0

0

1

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

1.5

0

0

Methyl methane-sulphonate

30 µg/mL

A

-

100

2

10

2

0

2

0

6

0

17

16

9

0

2

Methyl methane-sulphonate

40 µg/mL

A

-

100

3

14

3

0

2

5

9

0

25

24

9

0

0

 

Results: Experiment 2 - 22 hour treatment in the absence of S-9, 24 hour harvest

Concentration (µg/mL)

Replicate

Index (% of mean vehicle control cell count)

N

Number of structural aberrations

Frequency of structural aberrations (%)

% of cells with

Ctg

Ctb

Ctf

Csg

Csb

Csf

Cse

Other

With gaps

Without gaps

AE

ER

PP

0 (vehicle control)

A

98

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

102

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

260

A

91

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

105

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

521

A

108

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

106

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

1041

A

89

100

0

1

0

0

0

0

0

0

1

1

1

0

1

B

92

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

1

0

0

0

0

0

0

0

0.5

0.5

0

0.5

Methyl methane-sulphonate

30 µg/mL

A

-

100

4

9

1

0

0

0

6

0

15

14

0

0

0

Methyl methane-sulphonate

40 µg/mL

A

-

50

3

19

2

0

2

1

10

0

44

44

0

0

0

 

Results: Experiment 2 - 22 hour treatment in the absence of S-9, 48 hour harvest

Concentration (µg/mL)

Replicate

Index (% of mean vehicle control cell count)

N

Number of structural aberrations

Frequency of structural aberrations (%)

% of cells with

Ctg

Ctb

Ctf

Csg

Csb

Csf

Cse

Other

With gaps

Without gaps

AE

ER

PP

0 (vehicle control)

A

98

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

102

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

260

A

95

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

112

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

521

A

111

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

99

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

1041

A

101

100

0

0

0

0

0

0

0

0

0

0

0

0

0

B

115

100

0

0

0

0

0

0

0

0

0

0

0

0

0

Total

-

200

0

0

0

0

0

0

0

0

0

0

0

0

0

Methyl methane-sulphonate

20 µg/mL

A

-

100

0

2

0

0

0

0

0

0

2

2

1

0

0

Methyl methane-sulphonate

40 µg/mL

A

-

100

0

9

2

0

0

0

7

0

14

14

3

0

0

N:          Number of cells scored
Ctg:       Chromatid gaps
Ctb:       Chromatid breaks
Ctf:       Chromatid fragment
Csg:      Chromosome gap
Csb:      Chromosome break
Csf:       Chromosome fragment
Cse:      Chromosome exchange
AE:       Aneuploidy
ER:        Endoreduplication
PP:        Polyploidy

Applicant's summary and conclusion

Conclusions:
There was no evidence of clastogenicity with DMP Tech under the conditions of this study
Executive summary:

An in vitro chromosome aberration study was performed with DMP Tech (Dimethylolpropane tech) using Chinese Hamster Ovary (CHO) cells according to OECD test guideline 473. Testing was conducted in the absence and in the presence of S-9 for 6 hours with a harvest at 24 hours after the start of treatment, and in the absence of S-9 for 22 hours with a harvest at 24 and 48 hours. Toxicity was measured based on cell counts. No toxicity was observed in treated cultures. Testing was conducted in duplicate, except for the positive control where single cultures were employed. 100 metaphases were scored for each vehicle and positive control culture and for the highest three concentrations. There were no increases in the number of structural aberrations when compared to the concurrent vehicle control. It can be concluded, therefore, that there is no evidence for the clastogenicity of DMP Tech based on the results of this study.