[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

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:

1998-06-25 to 1998-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

[nitrilotris(methylene)]trisphosphonic acid, sodium salt

EC Number:

243-900-0

EC Name:

[nitrilotris(methylene)]trisphosphonic acid, sodium salt

Cas Number:

20592-85-2

Molecular formula:

General formula C3H12NO9P3.xNa where x=3-5 ATMP-3Na C3H9NNa3O9P3 ATMP-4Na C3H8NNa4O9P3 ATMP-5Na C3H7NNa5O9P3

IUPAC Name:

Sodium salt of [nitrilotris(methylene)]trisphosphonic acid (3-5Na:1)

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

up to 4400 µg active salt/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given in report

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

growth medium

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Controls 2

Untreated negative controls:

yes

Remarks:

growth medium

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (initial assay) 17.8 hours (confirmatory assay)
- Expression time (cells in growth medium): 16.8 hours (initial assay 2 hours (confirmatory assay)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (present for last 2 hours of incubation)
STAIN (for cytogenetic assays): 5% Giesma solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED:100 from each replicate culture where possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: assessment of percent confluence of cell monolayer and presence of mitotic or dead cells floating in medium.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


OTHER:

Evaluation criteria:

The test substance is considered positive for inducing chromosomal aberrations if there is significant dose-dependent increase (p<=0.01) in the number of cells with aberrations at one or more concentrations

Statistics:

Cochran-Armitage test for linear trend and Fisher's Exact test to compare percentage of cells with aberrations in treated cells with control results.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Non cytotoxic for 3h treatments. <500 µg/ml for continuous treatments without activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was measured at 9.0 (culture medium pH 8.5)
- Precipitation: no precipitation was observed
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES: no precipitate observed in the absence of cells at a concentration of 4400 ug/ml

COMPARISON WITH HISTORICAL CONTROL DATA: control values were within historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: dead monolayers and approximately 85% reduction in monolayer confluence reported at 4400 µg/ml. Unhealthy monolayers and approximately 70% reduction reported at 3080 µg/ml. Unhealthy monolayers and approximately15% or 45% reduction in monolayer confluence at 2160 µg/ml. In absence of metabolic activation no cytotoxicity was seen with 3 hr treatment. With continuous treatment cytotoxicity was induced.  The top dose scored (250 µg/ml) had a relative mitotic index of 60%.  The higher dose (500 µg/ml) had a relative mitotic index of <10%. 

Any other information on results incl. tables

Table 1: Results of chromosome analysis Experiment 1 (3 h treatment, 20 incubation) without activation (total count from 2 cultures / 200 cells)

-		Untreated	Solvent* Control	Positive Control	Low dose 1510 µg/ml	Mid dose 2160 µg/ml	Mid dose 3080   µg/ml	High dose 4400 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	3.0	1	52	3.5	0	3.0	0
Mitotic index (%)		14.8	20.8	NR	23.1	23.5	24.0	17.8
Polyploidy (%)		2.5	2.5	3.0	2.5	3.0	2.1	2.0
Endo reduplication (%)		2	1.5	1.0	2.0	1.5	0.5	2.5

 *Solvent control with water

NR not reported

 

 

Table 2: Results of chromosome analysis Experiment 2a (17.8 h treatment, 20 h incubation) without activation

-		Untreated	Solvent* Control	Positive Control	Low dose 31.3 µg/ml	Mid dose 62.6µg/ml	Mid dose 125 µg/ml	High dose 250 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	0	0	12.8	1.5	0	1.0	1.5
Mitotic index (%)		6.5	7.0	NR	4.0	6.9	4.0	4.2
Polyploidy (%)		9.5	0.5	2.5	1.5	0	0.5	0.5
Endo reduplication (%)		0	0	0	1.5	0	0	0

*Solvent control with water

NR not reported

 

Table 3: Results of chromosome analysis Experiment 1, (3 h treatment, 20 h incubation) with activation

-		Untreated	Solvent* Control	Positive Control	Low dose 1510 µg/ml	Mid dose 2160 µg/ml	Mid dose 3080 µg/ml	High dose 4400 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	1.5	1.0	60.0	1.0	8.0	1.5	1.0
Mitotic index (%)		16.0	19.9	NR	21.9	15.7	16.9	11.7
Polyploidy (%)		3.0	2.5	2.5	2.0	2.0	2.0	1.0
Endo reduplication (%)		1.5	0	1.5	1.0	6.0	0	0

*Solvent control with water

NR not reported

 

Table 4: Results of chromosome analysis Experiment 2, (3 h treatment, 20 h incubation) with activation

-		Untreated	Solvent* Control	Positive Control	Low dose 1510 µg/ml	Mid dose 2160 µg/ml	Mid dose 3080 µg/ml	High dose 4400 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	1.5	2.0	52	1.0	1.5	0.5	0.5
Mitotic index (%)		13.1	14.1	NR	12.8	13.8	13.3	13.3
Polyploidy (%)		2.0	1.5	2.0	2.0	3.0	2.0	2.5
Endo reduplication (%)		2.0	1.5	0.5	2.5	2.9	1.0	2.5

*Solvent control with water

NR not reported

Applicant's summary and conclusion

Conclusions:

ATMP-5Na has been tested for clastogenicity in vitro in Chinese hamster Ovary (CHO) cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (Covance Laboratories, 1998a). The test substance did not induce chromosome aberrations in vitro when tested up to cytotoxic concentration with or without metabolic activation. Positive, negative and solvent controls gave the expected results. It is concluded that ATMP-5Na does not cause chromosomal aberrations under the conditions of the test.