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Diss Factsheets

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)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines on Industrial Chemicals
Version / remarks:
1997
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate
EC Number:
701-216-4
Molecular formula:
DTPMP-5Na C9H23N3Na5O15P5 DTPMP-6Na C9H22N3Na6O15P5 DTPMP-7Na C9H21N3Na7O15P5
IUPAC Name:
octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}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
- Cells were purchased from Dainippon Pharmaceutical co.
- Cell suspension was mixed with one tenth volume of DMSO and then frozen and stored in liquid nitrogen. The suspended cells were used after thawing and cultivation through up to 5 passages
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
Remarks:
Mitomycin C was dissolved in saline
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
Remarks:
Benzo(a)pyrene was dissolved in Dimethyl Sulfoxide (DMSO)
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%
A cell with at least one structural chromosomal aberration was classified as aberrant cell. The number of aberrant cells was counted in two different ways, one includes cells with no aberration other than gaps and another excludes these types of cells.
Statistics:
When the test was positive the D20 values were calculated from the test results. The D20 value was the concentration (mg/ml) required to induce any aberration in 20% of metaphases.

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 (5-7Na) 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-dependant 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 (5-7Na) is positive for clastogenicity in vitro under the conditions of this test.