[ethylenebis[nitrilobis(methylene)]]tetrakisphosphonic acid, calcium sodium salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

May 08, 1981 to June 22, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline, with acceptable restrictions. The restrictions were that only 50 cells per animal were evaluated, and samples were collected only once. The study was conducted in compliance with GLP. Read-across to the registered substance is considered scientificaly justified.

Data source

Reference

Reference 1

Reference Type:

other: Study report

Title:

Unnamed

Year:

1981

Report date:

1981

Reference 2

Reference Type:

publication

Title:

Ethylenediaminetetra(methylenephosphonic acid): genotoxicity, biodistribution, and subchronic and chronic toxicity in rats

Author:

Calvin et al.

Year:

1988

Bibliographic source:

Food Chem Toxicol. Jul;26(7):601-10.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Only 50 cells/animal were evaluated instead of 100 cells. Animals were dosed for consecutive 5 days instead of single treatment and justification for same was not provided. Samples were collected only once instead of at least 2 times.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

EDTMP-H

IUPAC Name:

EDTMP-H

Constituent 2

Reference substance name:

[ethane-1,2-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid

EC Number:

215-851-5

EC Name:

[ethane-1,2-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid

Cas Number:

1429-50-1

Molecular formula:

C6H20N2O12P4

IUPAC Name:

{ethane-1,2-diylbis[nitrilobis(methylene)]}tetrakis(phosphonic acid)

Details on test material:

- Name of test material: Ethylenediamine tetraphosphoric acid (CC base)
- TSIN: E-0142
- Substance type: Pure active substance
- Physical state: White solid
- Stability under test conditions: Not reported
- Storage condition of test material: Not reported

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Outbred albino rats were obtained from Taconic farms
- Age at study initiation: Sexually mature
- Weight at study initiation: 125-200 g
- Assigned to test groups randomly: Yes, animals were randomized by weight
- Fasting period before study: Not reported
- Housing: Animals were housed individually.
- Diet: Food (Agway RMH 3000), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 7-9 d prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 69-75°F (mean)
- Humidity: 49-73% (mean)
- Air changes: Not reported
- Photoperiod: Not reported

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Amount of vehicle: 0.5 ml/100 g bw
- Concentration of test material in vehicle: 0.024, 0.08 and 0.24 mg/ml for 0.24, 0.80 and 2.40 mg/kg bw/d doses, respectively.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solution of test substance was prepared in corn oil. The dosing solution was prepared freshly prior to treatment.

DOSE VOLUME: 1 ml/100 g bw

Duration of treatment / exposure:

5d

Frequency of treatment:

Once daily

Post exposure period:

22-24 h after last treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
0.24, 0.8 and 2.4 mg/ kg bw/d
Basis:

No. of animals per sex per dose:

5 rats/sex/ group

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

- Positive control: Methylmethane sulfonate (MMS)
- Solvent used: Distilled water
- Route of administration: Oral gavage
- Doses / concentrations: 0.156 g/ kg bw/ d
- Dose volume: 1.5 ml/ 150 g bw
- Duration of dosing: Once daily for 5 d

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

SACRIFICE: Animals were sacrificed by cervical dislocation following CO2 anesthaesia after 2-4 h treatment with Colchicine.

TREATMENT AND SAMPLING: Animals were treated once daily for 5 consecutive days. Animals were treated with Colchicine (intraperitoneal; 1 mg/ kg) approx. 20 h after last treatment, to cause mitotic arrest of bone marrow cells. Bone marrow cells were collected 2-4 h after treatment with Colchicine. Bone marrow of both femurs was aspirated into 10cc syringe filled with prewarmed (37°C) 5 ml Hank’s balanced salt solution. After centrifugation (100x g), cells were suspended in 0.075 M KCl for 10 min at 37±1°C.

DETAILS OF SLIDE PREPARATION AND STAINING: 2-3 drops of cell suspension were placed onto microscopic slides and passed through flame. All slides were stained with Giemsa stain for 8 min and fixed with Carnoy’s fixative. Slides were washed in water (1 min) then with acetone (10-20 sec), then in acetone-xylene (1:1 v/ v; 10-20 sec) and finally in xylene for 5 min. Permount was used as the mounting and all slides were placed on slide warmer at 30-35°C for minimum 2 h before scoring.

METHOD OF ANALYSIS: 50 metaphase/ animal were analyzed. Cytogenetic abnormalities such as deletions, exchanges, rings, gaps and breaks were scored and mitotic index of each animal was determined. The vernier location for each cell scored was recorded.

Statistics:

Standard deviation (SD) and standard error (SE) was calculated.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

DETAILS ON CLINICAL SIGNS AND SYMPTOMS:

- Clinical signs: Animals treated with test substance revealed no signs of toxicity.

- Mortality: No mortality was observed in negative, vehicle and test substance treated groups.

RESULTS OF POSITIVE CONTROL:

- Clinical signs: Some of positive control group animals revealed signs of ataxia, inactivity, lethargy, weakness and hypersensitivity.

- Mortality: 4 animals of positive control group died during study.

Table 1. Bone marrow aberrations after treatment with Ethylenediamine tetraphosphoric acid and controls (Study # 25691)

Treatment	% of Total Cells Analyzed
	Total aberrations (including gaps)		Aberrations excluding gaps
	Male	Female	Male	Female
Distilled water	0.6	2.8	0	0.4
Corn oil	4.0	7.2	0	1.6
MMS(0.156 g/ kg bw)	18.0	38.5	9.0	15.4
Test substance (0.24 g/ kg bw)	3.6	4.4	0	0.4
Test substance (0.80 g/ kg bw)	2.0	4.8	0.4	0.8
Test substance (2.40 g/ kg bw)	4.0	2.8	0.4	0

MMS= Methylmethane sulfonate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Ethylenediamine tetraphosphoric acid (CC base) has been tested in a chromosome aberration assay conducted according to a protocol that is similar to OECD 475 and under GLP conditions. The substance was administered orally at 0.24, 0.80 and 2.40 mg/kg bw/d to albino Sprague Dawley rats for 5 days. No test substance treatment related mortalities were observed. Appropriate solvent (corn oil), negative (water) and positive controls were included and gave expected results. The test substance did not induce any chromosomal damage in the bone marrow cells of the treated rats. It is concluded that EDTMP-H is negative for the induction of chromosome aberrations in rat bone marrow under the conditions of the study.