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Genetic toxicity: in vitro

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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:
2010-02-22 to 2010-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP study according to recent OECD TG

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)
Version / remarks:
July 21, 1997
Deviations:
yes
Remarks:
The study was performed according to the corresponding study plan except a supplemental pre-experiment on cytotoxicity with S9-mix.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The study was performed according to the corresponding study plan except a supplemental pre-experiment on cytotoxicity with S9-mix
GLP compliance:
yes (incl. certificate)
Remarks:
certificate attached to full study report
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: D-MEM with high glucose, GlutaMax and sodiumpyruvate, supplemented with 10% FBS and penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Short treatment: 4h with and without S9: 0, 60, 600, 1600, and 5000µg/mL
Long treatment: 24h without S9: 10, 30, 60, and 120µg/mL
Vehicle / solvent:
VEHICLES USED:
- Concentrated stock solutions of Graphite were prepared using phosphate buffer (pH 7.4) with 100µg/mL soy lecithin
- For destruction of agglomerates energy input by ultrasound was used
- Stability of the stock solutions was demonstrated by physicochemical characterisation, comprising particle size distribution, zetapotential and pH

JUSTIFICATION FOR CHOICE OF VEHICLE
- Non-toxic natural emulsifier to enable wetting of the highly hydrophobic graphite particle surface
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Incubation media
Negative solvent / vehicle controls:
yes
Remarks:
Incubation media supplemented with vehicle
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: without S9
Untreated negative controls:
yes
Remarks:
Incubation media
Negative solvent / vehicle controls:
yes
Remarks:
Incubation media supplemented with vehicle
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium with vehicle

DURATION
- Exposure duration: 4h (short term) and 24h (long term)
- Expression time (cells in growth medium): 24h+4h and 24h+24h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Conventional Giemsa stain

NUMBER OF REPLICATIONS: Duplicate cultures per treatment group

NUMBER OF CELLS EVALUATED: 200 metaphases per treatment (100 from each culture A and B)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: mitotic index
Evaluation criteria:
- Evaluation by comparing treatment groups with positive, vehicle, concurrent and historical negative controls
- Main aspect is biological relevance
- Experimental unit is the cell, therefore the percentage of cells with structural chromosome aberrations has more impact than the total number of aberrations in 200 metaphases
- Cytotoxicity of the test item as well as extreme alterations of culture conditions (pH, osmality) by the test item are also considered
- Statistical analysis serves as aid for determining a positive respons
- A result is positive, if the number of aberrant cells falls within the range of concurrent positive controls or if there is a dose-related increase in the percentages of aberrant cells in comparison to the concurent negative/vehicle controls
- The test item is also considered to be positive if a reproducible increase in the number of cells with aberrations occurs for at least one test condition
- An increase in polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations
Statistics:
- chi^2 or Fisher's exact test may be used as an aid in evaluating the test results, but should not be the only basis for determining a positive response

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: See additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH for test item containing media as compared to the negative and vehicle control
- Effects of osmolality: Slight changes in osmolality, which were still in the physiological range for all treatment media
- Water solubility: In addition, turbidity/precipitation in the test item solutions was measured by applying the OD600 method of an Eppendorf BioPhotometer (Hamburg, Germany). The samples were agitated and turbidity was measured at a wavelength of 595 nm. There was a linear, concentration-dependent increase in OD600, indicating low solubility (Expanded Graphite Powder is water insoluble and needed a special method to prepare the treatment media) and thus precipitation. Turbidity was already noted at 25 µg/ml Expanded Graphite Powder [91.86 Formazine Attenuation Units (FAU)].

RANGE-FINDING/SCREENING STUDIES:
- Cytotoxicity of the test item was evaluated in pre-experiments both with and without metabolic activation, using a broad range of concentrations (10, 25, 50, 100, 250, 500, 1000, 2500, and 5000 µg/ml) and the mitotic index (M.I.) as endpoint.

COMPARISON WITH HISTORICAL CONTROL DATA: Observed low aberration frequencies were in the range of historical negative controls

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The observed cytotoxicity of the test item more likely represents particle-like effects or mechanical effects due to the high concentrations used and perhaps the occurrence of particle agglomerates in aqueous suspension.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

TABLE 1: Aberration Frequencies of Cells and % Metaphases ( means of Exp. A and B ):

 

4h / -S9-mix

4h / +S9-mix

24h / –S9-mix

Treatment

% AC

 

% AC

 

% AC

 

 

+g

-g

%M

+g

-g

%M

+g

-g

%M

Negative control

2.5

0.0

100

1.5

0.5

100

0.5

0.0

100

Vehicle control

1.5

0.0

90

2.0

0.0

100

1.0

0.0

111

EMS: 200 µg/ml

16.5

9.0

76

-

-

-

-

-

-

EMS: 100 µg/ml

-

-

-

-

-

-

44.5

35.0

58

CP:  2.5 µg/ml

-

-

-

66.0

54.0

91

-

-

-

Test item

 

 

 

 

 

 

 

 

 

10 µg/ml

-

-

-

-

-

-

1.5

0.0

93

30 µg/ml

-

-

-

-

-

-

1.0

0.0

78

60 µg/ml

2.0

0.5

94

1.0

0.5

100

1.0

0.0

66

120 µg/ml

-

-

-

-

-

-

2.5

0.5

37

600 µg/ml

2.0

0.5

78

2.5

0.5

105

-

-

-

 1600 µg/ml

3.0

0.5

68

3.5

0.0

101

-

-

-

5000 µg/ml

2.5

0.5

56

2.5

0.5

96

-

-

-

 % AC = % aberrant cells; % M = M.I. in percent of the negative control (mean value of % M of cultures A and B); + g = with gaps; -g = without gaps; - = concentration not used; M.I. see Tables 2 and 3.

 

TABLE 2: Mitotic indices ( M.I. ), main experiments, 4 and 24 h treatment, without S9-mix

Replicate No.:A and B
Exposure to the test item:4 h and 24 h without S9-mix
Fixation time:24 hafter treatment start
Evaluated cells: 1000 per culture

 

4 hours

24 hours

Treatment group

A

B

A

B

 

M.I.

%

M.I.

%

M.I.

%

M.I.

%

Negative control

9.0

100

9.5

100

10.4

100

9.4

100

Vehicle control

8.1

90

8.6

91

10.9

105

11.0

117

Positive control
EMS:
   50µg/ml

-

-

-

-

6.2

60

4.4

47

Positive control
EMS:
 100µg/ml

-

-

-

-

5.1

49

6.3

67

Positive control
EMS:
 150µg/ml

7.6

84

9.8

103

-

-

-

-

Positive control
EMS:
 200µg/ml

7.2

80

6.9

73

-

-

-

-

test item:    10µg/ml

-

-

-

-

9.2

88

9.3

99

test item:    30µg/ml

-

-

-

-

8.0

77

7.4

79

test item:    60µg/ml

8.6

96

8.8

93

6.8

65

6.3

67

test item:  120µg/ml

-

-

-

-

4.1

39

3.3

35

test item:  600µg/ml

7.6

84

6.8

72

-

-

-

-

test item:1600µg/ml

7.3

81

5.2

55

-

-

-

-

test item:5000µg/ml

5.4

60

5.0

53

-

-

-

-

M.I.       =   (number of metaphases/number of cells counted) x 100 
%
          =   percent of control
A = replicate A
B = replicate B
-
 = not determined


TABLE 3: Mitotic indicex ( M.I. ), main experiment, 4 h treatment, with S9-mix

Replicate No.:A and B
Exposure to the test item:4 h with S9-mix
Fixation time:24 hafter treatment start
Evaluated cells: 1000 per culture

 

4 hours

Treatment group

A

B

 

M.I.

%

M.I.

%

Negative control

7.9

100

8.2

100

Vehicle control

8.0

101

8.1

99

Positive control
CP:2.0µg/ml

8.2

104

8.3

101

Positive control
CP:2.5µg/ml

7.5

95

7.1

87

Test item:     60µg/ml

8.2

104

7.9

96

Test item:   600µg/ml

8.4

106

8.5

104

Test item: 1600µg/ml

8.2

104

8.1

99

Test item: 5000µg/ml

7.8

99

7.7

94

M.I.       =   (number of metaphases/number of cells counted) x 100 
%
          =   percent of control
A = replicate A
 
B = replicate B

OBSERVED CHROMOSOMAL ABERRATION TYPES:

Historical negative controls: g, G,b, B, ex, e, py
Concurrent negative and vehicle controls:
 g, G, b, py

Test item treated cells:
 
Short treatment:
 g, G, b, B, ex, py, e
Long term treatment:
 g, B, py, e

KEY TO ABERRATIONS

The criteria for scoring aberration are based on the classification of Buckton and Evans (1973), ISCN (1985), and Savage (1976 and 1983). Standard forms are used to record aberrations:

Gaps:
ctg*
  
(g)**chromatid type
csg
   (G)  isochromatid / chromosome type

Deletions / Breaks:

Chromatid type
:
ctb
      (b)                  chromatid break
ct_min (b)
                  chromatid minute

Chromosome type:
f
         (B)                  acentric fragment
dmin
   (B)                  double minute
su
       (B)                  sister union (isochromatid break)
nud
     (B)                  non-union distal (isochromatid break)
nup
     (B)                  non-union proximal (isochromatid break)

Exchanges:

Chromatid type:

qr        (ex)     quadriradial
tr
         (ex)     triradial
ctr
       (ex)     chromatid ring
ct_inv
  (ex)     chromatid inversion
cte      (ex)     chromatid intrachange

Chromosome type:

dic(+f)           (EX)     dicentric (+ fragment)
csr(+f)(EX)
     centric ring (+ fragment)
cx
       (EX)     complex exchange

Others:

                   (at)     atypical chromosome
Endo
            (e)      endoreduplication
mabs
           (ma)   multiple aberrations (10 or more structural aberrations, gaps included)
Hyper
                     hyperploidy
Poly
             (py)    polyploidy

*Abbreviations in bold letters are used in the Microptic system for definition of chromosomal aberrations. **Abbreviations in parentheses are used in summing up tables in the raw data and the tables of the final report as well as in the text of the final report.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation (S9)

Due to the low aberration frequencies in Expanded Graphite Powder-treated cultures, which are in the range of historical negative controls, the test item Graphite is considered not to induce structural chromosomal aberrations in cultured mammalian somatic cells under the test conditions used.
Executive summary:

Graphite Powder was tested for its ability to induce structural (and numerical) chromosomal aberrations in V79 cells. The study was conducted in compliance with the OECD Guideline for the Testing of Chemicals No. 473 (Genetic Toxicology: In Vitro Mammalian Chromosome Aberration Test, July 21, 1997) and the Commission Regulation (EC) No. 440/2008, Part B.10. (Mutagenicity – In Vitro Mammalian Chromosome Aberration Test, May 30, 2008).

Expanded Graphite Powder did not mediate marked changes in osmolality and pH of the incubation media. The slight decrease in osmolality, which was observed for the test item-containing incubation media, compared to the negative control medium, was mainly due to addition of the vehicle component phosphate buffer (pH 7.4) with 100 µg/ml soy lecithin, used to suspend the hydrophobic test item.

Negative controls exhibited spontaneous aberration frequencies within the normal range for V79 cells. Positive controls (without S9 -mix and CP with S9 -mix) markedly increased the frequency of aberrant cells, with aberration frequencies clearly exceeding 5 % as the defined lower limit for positive control substances. Test performance and activity of the metabolizing system were thus satisfactory. Observed cytotoxicity of the test item is likely to represent a particle- or mechanical effect due to high concentrations used and perhaps the occurrence of particle agglomerates in aqueous suspension. After incubation of the test-item suspension together with S9 -mix nearly no cytotoxicity was observed. This might indicate better suspension of the particles due to the high protein content of the S9 -mix or perhaps protein-dependent masking of reactive groups on the particle surface.

Graphite Powder, under all treatment modalities, did not increase significantly the frequency of aberrant cells, as compared to the respective negative controls. Besides some gaps, which were also evident in the negative and solvent control cultures, a sum of 6 chromatid and chromosome breaks, 5 exchanges, and 3 endoreduplications were noted in all 3 main experiments, without clear concentration-dependency. The resulting frequencies of aberrant cells (without gaps) in no case exceeded 5 % as defined lower limit for positive substances and all aberration frequencies fell within the range of the historical negative controls.