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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
Study period:
15-Jun-2001 to 18-Sep-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines on Industrial Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
[[(phosphonomethyl)imino]bis[(ethylenenitrilo)bis(methylene)]]tetrakisphosphonic acid, sodium salt
EC Number:
244-751-4
EC Name:
[[(phosphonomethyl)imino]bis[(ethylenenitrilo)bis(methylene)]]tetrakisphosphonic acid, sodium salt
Cas Number:
22042-96-2
Molecular formula:
C9H28N3O15P5.xNa
IUPAC Name:
sodium hydrogen [10,10-dihydroxy-10-oxido-2,5,8-tris(phosphonomethyl)-2,5,8-triaza-10-phosphadec-1-yl]phosphonate
Test material form:
solid - liquid: aqueous solution

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: CHL/IU (originally derived from female Chinese hamster lung)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimum essential medium
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and 5,6-benzoflavone-induced male rat liver S9
Test concentrations with justification for top dose:
625-5000 µg mixed sodium salts/ml (pulse treatment)
150-3000 µg mixed sodium salts/ml (24-hour continuous treatment)
50-400 µg mixed sodium salts/ml (48-hour continuous treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: solubility of test substance in water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9, 0.1 µg/ml (pulse treatment), 0.03 µg/ml (continuous treatment)
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9, 15 µg/ml (pulse treatment)
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: pulse treatment 6 hours; continuous treatment, 24 or 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): pulse treatment 24 hours; continuous treatment 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.1 µg/ml, 2 hours
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 100 cells /culture, 2 cultures/treatment

DETERMINATION OF CYTOTOXICITY
- Method: other: cell density after crystal violet staining

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: mitotic index
Evaluation criteria:
Negative: both the incidence of aberrant cells (excluding gaps) and that of numerical aberrations <5%
Inconclusive: either of these incidences is 5-10%
Positive: either of these incidences >10%
Statistics:
None

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
pulse treatment ( 6 and 24 hour exposure)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=2500 µg mixed sodium salts/ml (pulse treatment),
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
48 hour treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=50 µg mixed sodium salts/ml (48-hour continuous treatment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- No, but cell survival was evaluated at 156-5000 µg mixed sodium salts/ml (pulse treatment, 24-hour continuous treatment and 48-hour continuous treatment) and the data used to determine the concentrations that were to be evaluated for chromosome aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cell survival:
- Pulse treatment -S9, µg mixed sodium salts/ml: 156 (109%), 313 (109%), 625 (97%), 1250 (96%), 2500 (76%), 5000 (58%)
- Pulse treatment +S9, µg mixed sodium salts/ml: 156 (98%), 313 (104%), 625 (103%), 1250 (108%), 2500 (102%), 5000 (77%)
- 24-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (115%), 313 (81%), 625 (47%), 1250 (37%), 2500 (19%), 5000 (0%)
- 48-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (50%), 313 (25%), 625 (7%), 1250 (5%), 2500 (1%), 5000 (0%)

Any other information on results incl. tables

Full details of the results from the 48 hours treatment (result reported as positive) are not presented in the study report. A graph of the results indicates that the incidence of cells with structural aberrations was 2.5% at 50 µg/ml; 5% at 100; 15% at 150; 37% at 200 and 49% at 300 µg/ml.

Table 1 Chromosome aberration test - treatment time 6 hours

Concentration µg/ml

+/- S9 mix

No. of cells analyzed

Chromatid breaks

Chromatid exchanges

No. of cells with aberration (%)

No. of cells with gaps

Cell growth index (%)

Polyploid cells

Control

-

200

0

0

0

1

100

0

625

-

200

1

0

1

0

98

0

1250

-

200

3

1

4

0

91

0

2500

-

200

6

1

7

4

71

2

5000

-

200

9

1

9

3

41

0

Positive control

-

200

54

121

139

1

-

0

Control

+

200

0

0

1

0

100

0

625

+

200

0

2

2

1

91

0

1250

+

200

0

1

1

0

95

0

2500

+

200

3

3

4

0

99

0

5000

+

200

1

0

1

0

67

0

Positive control

+

200

7

42

46

1

-

0

 

Chromosome aberration test - treatment time 24 hours

Concentration µg/ml

+/- S9 mix

No. of cells analyzed

Chromatid breaks

Chromatid exchanges

No. of cells with aberration (%)

No. of cells with gaps

Cell growth index (%)

Polyploid cells

Control

-

200

2

0

2

1

100

0

4.7

-

200

1

1

2

1

101

0

9.4

-

200

2

0

2

0

102

0

18.8

-

200

4

0

4

1

104

0

37.5

-

200

3

2

5

0

107

0

75

-

200

2

1

3

3

103

0

150

-

200

8

0

8

2

113

0

300

-

-

TOXIC

Positive control

-

200

30

30

58

0

-

0

Control

+

200

2

0

2

0

100

0

50

+

200

4

1

5

0

99

0

100

+

200

7

4

11

2

68

0

150

+

200

31

9

34

0

57

0

200

+

200

67

9

74

1

43

0

300

+

200

87

10

97

2

20

0

400

+

-

TOXIC

Positive control

+

200

47

56

86

0

-

0

 

Applicant's summary and conclusion

Conclusions:
DTPMP-7Na has been tested for clastogenicity in a valid in vitro mammalian chromosome aberration study in mammalian cell line CHL/IU with and without metabolic activation, conducted according to OECD Test Guideline 473 and in compliance with GLP. A dose-related increase in the number of cells with aberrations was observed after 48 hours treatment, without metabolic activation. No dose-depended increase in the number of cells with aberrations was observed after 6 or 24 hours treatment, with and without metabolic activation. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-7Na is positive for clastogenicity in vitro under the conditions of this test.