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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-04-18 to 2011-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
EC Number: 939-946-2

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were obtained from a healthy donor not receiving medication. For this study, blood was collected from a female donor (37 years old).
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 h after blood collection. If necessary, the blood was stored before use at 4 °C.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
between 15.4 and 2370 microgram/mL
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure.
Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks. The culture medium was DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1; already supplemented with 15 mM HEPES and 200 mM L-glutamine. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 Ng/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin (PHA, final concentration 3 Ng/mL, 10 % FBS (fetal bovine serum), the anticoagulant heparin.
About 72 h after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 NL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described.
The "saline G" solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
glucose•H2O 1100 mg
Na2HPO4•2H20 192 mg
KH2PO4 150 mg
pH was adjusted to 7.2
After washing the cells were re-suspended in complete culture medium and cultured until preparation. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
Evaluation criteria:
The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for three values in the presence of S9 mix, where 50 metaphases were scored. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.
A test item is classified as non-mutagenic if:
the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data
no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data
and either a concentration-related or a significant increase of the number of structuralchromosome aberrations is observed.
However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05)

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
induced structural chromosomal aberrations in human lymphocytes in vitro
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
51.2% at highest evaluated concentration in absence of S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures

Any other information on results incl. tables

TABLE 1     Doses applied in the Chromosome Aberration Assay with Test Item

Preparation
interval

Exposure
period

Concentrations in µg/mL

Without S9 mix

 

22 hrs

 4 hrs

15.4

26.9

47.2

82.5

144.4

252.7P

442.9P

773.9P

1354.3P

2370.0P

With S9 mix

22 hrs

 4 hrs

15.4

26.9

47.2

82.5

144.4

252.7P

442.9P

773.9P

1354.3P

2370.0P

        Evaluated experimental points are shown in bold characters
P
       Precipitation was observed at the end of treatment

 

TABLE 2     Summary of Results

Summary of results of the chromosomal aberration study with test item

Preparation

Test item

Mitotic indices

Aberrant cells

interval

concentration

in %

in %

 

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix

22 hrs

Solvent control1

100.0

2.5

2.0

0.0

 

Positive control2

62.3

8.5

8.0S

0.5

 

144.4

78.8

2.5

2.5

0.5

 

252.7P

72.4

3.5

2.5

0.0

 

1354.3P

51.2

10.0

10.0S

2.0

Exposure period 4 hrs with S9 mix

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

Positive control3

65.2

12.5

12.5S

2.0

 

15.4

72.8

11.5

11.0S

0.5

 

26.9#

89.8

45.0

44.0S

0.0

 

47.2

82.2

16.0

16.0S

2.0

 

82.5#

102.9

52.0

52.0S

5.0

 

144.4#

101.6

61.0

61.0S

4.0

*      Including cells carrying exchanges

#       Evaluation of 50 metaphases per culture

P       Precipitation occurred at the end of treatment

S       Aberration frequency statistically significant higher than corresponding control values

1       DMSO       0.5 % (v/v)

2       EMS      825.0 µg/mL

3       CPA           7.5 µg/mL

TABLE 3      Toxicity – Main Experiment (Cytotoxicity of Test Item to the Cultures of Human Lymphocytes)

In the Main Experiment the mitotic index in two cultures (1000 cells per culture) was determined.

Concentration
(µg/mL)

Exposure time

Preparation interval

Mitotic cells
per 1000 cells*

% of
solvent control

Without S9 mix

Solvent control

4 hrs

22 hrs

18.9

100.0

15.4

4 hrs

22 hrs

19.7

104.2

26.9

4 hrs

22 hrs

18.4

97.3

47.2

4 hrs

22 hrs

17.7

93.9

82.5

4 hrs

22 hrs

16.5

87.3

144.4

4 hrs

22 hrs

14.9

78.8

252.7P

4 hrs

22 hrs

13.7

72.4

442.9P

4 hrs

22 hrs

12.9

68.4

773.9P

4 hrs

22 hrs

9.3

49.3

1354.3P

4 hrs

22 hrs

9.7

51.2

2370.0P

4 hrs

22 hrs

7.7

40.6

With S9 mix

Solvent control

4 hrs

22 hrs

19.1

100.0

15.4

4 hrs

22 hrs

13.9

72.8

26.9

4 hrs

22 hrs

17.2

89.8

47.2

4 hrs

22 hrs

15.7

82.2

82.5

4 hrs

22 hrs

19.7

102.9

144.4

4 hrs

22 hrs

19.4

101.6

252.7P

4 hrs

22 hrs

11.5

60.2

442.9P

4 hrs

22 hrs

13.9

72.8

773.9P

4 hrs

22 hrs

0.0

0.0

1354.3P

4 hrs

22 hrs

4.9

25.7

2370.0P

4 hrs

22 hrs

0.0

0.0

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*    Mean value of two cultures in %
P
    Precipitation occurred at the end of treatment

TABLE 4               Main Experiment-Mitotic Index;
                              (Preparation Interval 22 hrs with and without S9 Mix)

Treatment

Conc.

S9

Exposure

Polyploid

Mitotic Indices*

group

per mL

mix

period/

cells

Absolute

Mean

%**

 

 

 

Recovery (hrs)

 

1

2

 

 

Solv. control#

      0.5  %

-

4 / 18 hrs

No polyploidies observed

18.6

19.1

18.9

100.0

Pos. control##

 825.0       µg

-

4 / 18 hrs

"

11.5

12.0

11.8

62.3

Test item

 144.4       µg

-

4 / 18 hrs

"

14.8

14.9

14.9

78.8

"

 252.7       µg

-

4 / 18 hrs

"

14.7

12.6

13.7

72.4

"

1354.3       µg

-

4 / 18 hrs

"

11.1

8.2

9.7

51.2

Solv. control#

      0.5  %

+

4 / 18 hrs

No polyploidies observed

16.8

21.4

19.1

100.0

Pos. control###

      7.5       µg

+

4 / 18 hrs

"

10.8

14.1

12.5

65.2

Test item

    15.4       µg

+

4 / 18 hrs

"

11.6

16.2

13.9

72.8

"

    26.9       µg

+

4 / 18 hrs

"

14.1

20.2

17.2

89.8

"

    47.2       µg

+

4 / 18 hrs

15.5

15.9

15.7

82.2

"

    82.5       µg

+

4 / 18 hrs

"

19.7

19.6

19.7

102.9

"

 144.4       µg

+

4 / 18 hrs

"

20.8

18.0

19.4

101.6

*       The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

**      For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls

#           DMSO

##         EMS

###       CPA

TABLE 5       Structural Chromosome Aberrations Main Experiment;
                       (Preparation Interval 22 hrs without S9 Mix:
                       Exposure Period 4 hrs)

Slide

Cells

% Aberrant cells

Aberrations

 

no.

scored

incl.

excl.

carrying ex-changes

Gaps

Chromatid type

Chromosome type

Other

 

 

 

gaps*

gaps*

 

g

ig

b

f

d

ex

ib

if

id

cx

ma

cd

 

 

Solvent control: DMSO 0.5 %

Without S9 mix

1

100

 

 

 

0

0

2

0

0

0

0

0

0

0

0

0

2

100

 

 

 

1

0

2

0

0

0

0

0

0

0

0

0

1 + 2

200

2.5

2.0

0.0

1

0

4

0

0

0

0

0

0

0

0

0

Positive control: EMS 825.0 µg / mL

 

1

100

 

 

 

0

0

6

1

0

0

0

1

0

0

0

0

2

100

 

 

 

1

0

7

2

0

1

0

0

0

0

0

0

1 + 2

200

8.5

8.0

0.5

1

0

13

3

0

1

0

1

0

0

0

0

Test item: 144.4 µg / mL

 

1

100

 

 

 

0

0

2

0

0

0

0

0

0

0

0

0

2

100

 

 

 

0

0

1

0

0

1

0

1

0

0

0

0

1 + 2

200

2.5

2.5

0.5

0

0

3

0

0

1

0

1

0

0

0

0

Test item: 252.7 µg / mL

 

1

100

 

 

 

1

0

3

0

0

0

0

0

0

0

0

0

2

100

 

 

 

1

0

2

0

0

0

0

0

0

0

0

0

1 + 2

200

3.5

2.5

0.0

2

0

5

0

0

0

0

0

0

0

0

0

Test item: 1354.3 µg / mL

 

1

100

 

 

 

0

0

10

0

0

3

0

0

0

0

1

0

2

100

 

 

 

0

0

7

0

0

2

0

2

0

0

0

0

1 + 2

200

10.0

10.0

2.0

0

0

17

0

0

5

0

2

0

0

1

0

*      Including cells carrying exchanges

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

TABLE 6     Structural Chromosome Aberrations Main Experiment;
                       (Preparation Interval 22 hrs with S9 Mix: Exposure Period 4 hrs)

Slide

Cells

% Aberrant cells

Aberrations

 

no.

scored

incl.

excl.

carrying ex-changes

Gaps

Chromatid type

Chromosome type

Other

 

 

 

gaps*

gaps*

 

g

ig

b

f

d

ex

ib

if

id

cx

ma

cd

 

 

Solvent control: DMSO 0.5 %

Without S9 mix

1

100

 

 

 

0

0

0

0

0

0

0

0

0

0

0

0

2

100

 

 

 

0

0

2

0

0

0

0

0

0

0

0

0

1 + 2

200

1.0

1.0

0.0

0

0

2

0

0

0

0

0

0

0

0

0

Positive control: CPA 7.5 µg / mL

 

1

100

 

 

 

0

0

12

0

0

1

1

0

0

0

0

0

2

100

 

 

 

0

0

15

0

0

3

1

0

0

0

0

0

1 + 2

200

12.5

12.5

2.0

0

0

27

0

0

4

2

0

0

0

0

0

Test item: 15.4 µg / mL

 

1

100

 

 

 

1

0

11

0

0

0

1

0

0

0

0

0

2

100

 

 

 

0

0

9

0

0

1

0

2

0

0

1

0

1 + 2

200

11.5

11.0

0.5

1

0

20

0

0

1

1

2

0

0

1

0

Test item: 26.9 µg / mL**

 

1

50

 

 

 

1

0

21

1

0

0

0

0

0

0

0

0

2

50

 

 

 

0

0

38

4

0

0

2

0

0

0

7

0

1 + 2

100

45.0

44.0

0.0

1

0

59

5

0

0

2

0

0

0

7

0

Test item: 47.2 µg / mL

 

1

100

 

 

 

1

0

14

1

0

1

0

1

0

0

0

0

2

100

 

 

 

2

0

13

0

0

3

0

0

0

0

0

0

1 + 2

200

16.0

16.0

2.0

3

0

27

1

0

4

0

1

0

0

0

0

Test item: 82.5 µg / mL**

 

1

50

 

 

 

2

0

41

0

0

3

0

0

0

0

4

0

2

50

 

 

 

2

0

31

0

0

3

1

0

0

0

3

0

1 + 2

100

52.0

52.0

5.0

4

0

72

0

0

6

1

0

0

0

7

0

Test item: 144.4 µg / mL**

 

1

50

 

 

 

0

0

53

3

0

2

2

2

0

0

10

0

2

50

 

 

 

0

0

30

1

0

2

0

0

0

0

6

0

1 + 2

100

61.0

61.0

4.0

0

0

83

4

0

4

2

2

0

0

16

0

*      Including cells carrying exchanges

**    Evaluation of 50 metaphases per culture

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

TABLE 7     Biometry

Statistical significance at the five per cent level (p < 0.05) for aberration frequency was evaluated by means of the Fisher’s exact test. Evaluation was performed only for cells carrying aberrations excluding gaps.

Biometry of the Main Experiment

Test group versus
solvent control

Preparation
interval

Exposure
period

S9 mix

p-value

Test group

144.4µg/mL

22 hrs

4 hrs

-

0.376

"

252.7µg/mL

22 hrs

4 hrs

-

0.376

"

1354.3µg/mL

22 hrs

4 hrs

-

0.003S

"

15.4µg/mL

22 hrs

4 hrs

+

< 0.001S

"

26.9 µg/mL

22 hrs

4 hrs

+

< 0.001S

"

47.2 µg/mL

22 hrs

4 hrs

+

< 0.001S

"

82.5 µg/mL

22 hrs

4 hrs

+

< 0.001S

"

144.4 µg/mL

22 hrs

4 hrs

+

< 0.001S

Positive control versus
solvent control

 

 

 

 

EMS

825.0 µg/mL

22 hrs

4 hrs

-

0.003S

CPA

7.5 µg/mL

22 hrs

4 hrs

+

< 0.001S

n.c.  Not calculated as the aberration rate is equal or lower than the control rate
S
       Aberration rate is statistically significant higher than the control rate

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation, when tested up to cytotoxic and/or precipitating or the highest evaluable concentration.
The study is considered to be relevant, reliable, adequate for risk assessment, and adequate for classification purposes.
Executive summary:

Study Design:

This in vitro assay was performed to assess the potential of the test item to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for three values in the presence of S9 mix, where 50 metaphases were scored. The highest applied concentration in the main study (2370.0 Ng/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (92.9 %) of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation and in accordance with OECD Guideline 473.

Results:

In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration.

In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.

Clastogenicity was observed in the absence of S9 mix after treatment with 1354.3 Ng/mL (10.0 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment with all evaluated concentrations. All values for percent aberrant cells (excluding gaps) are statistically significant and above the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Conclusion:

Under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic and/or precipitating or the highest evaluable concentration