Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7 Na:1)

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

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

Test material information

Constituent 1

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

Controls

Controls 1

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

Controls 2

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 results

Test results 1

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

Test results 2

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.