Nitrilotriacetic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well-documented publication which meets basic scientific principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Principles of method if other than guideline:

Trilon AS (nitrilotriacetic acid ; NTA) is intended to serve as positive control substance for a screening of kidney toxicity.

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Test material

Test material information

Constituent 1

Reference substance name:

Trilon AS

IUPAC Name:

Trilon AS

Constituent 2

Reference substance name:

Nitrilotriacetic acid

EC Number:

205-355-7

EC Name:

Nitrilotriacetic acid

Cas Number:

139-13-9

Molecular formula:

C6H9NO6

IUPAC Name:

2-[bis(carboxymethyl)amino]acetic acid

Details on test material:

Chemical name: nitrilotriacetic acid
Degree of purity: > 98%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Only animals free from clinical signs of disease were used for the study. The rats were identified clearly by tattooing of the respective animal number into the ears.
During the test, the rats were housed singly in type DK III stainless steel wire mesh cages supplied by BECKER & Co., Castrop-Rauxel, FRG (floor area about 800 cm2). The animal cages were placed on the racks in such a way that uniform test conditions (air inflow/air outflow/light) were guaranteed. The animals were housed in a fully air-conditioned room. Central air conditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70 % for relative humidity. The day/night rhythm was 12 hours (12 hours light from 6.00 a.m. - 6.00 p.m., 12 hours dark from 6.00 p.m. - 6.00 a.m.).
The animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin/ammonia-based terminal disinfector) before the start of the study. The floor was cleaned twice a week and the walls were cleaned once a week. The cleansing liquid used in each case was water containing about 0.1 % Incidin perfektO (supplied by Henkel, Duesseldorf, FRG) .
The food received by the animals was ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland, which was available ad libitum, as was drinking water.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

For the preparation of the doses, the test substance was weighed out in a calibrated beaker, diluted to the appropriate volume with 0 .5% CMC (carboxymethyl cellulose and mixed thoroughly using a high speed sonicator. During the administration the doses were kept homogeneous by using a magnetic stirrer. The mixtures were prepared twice a week. The stability of the test substance in the vehicle over 4 days at room temperature and the homogeneous distribution was proven in a previous study (Project No.: 25S0109/94003). The correctness of the concentrations was checked at all concentrations at the start of the administration period.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

3 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
150, 500, 1500 mg/kg
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Trilon AS (nitrilotriacetic acid; NTA) is intended to serve as positive control substance for a screening of kidney toxicity. The aim of the present study was to examine if the strain of rats used at BASF Toxicology exhibits signs of nephrotoxicity after repeated administration of the test substance. Based upon the low acute toxicity of the test substance, following doses were chosen for the present study: 150 ; 500 and 1,500 mg/kg body weight.

Examinations

Observations and examinations performed and frequency:

Food consumption: The food consumption over a period of 7 days was determined weekly and calculated as mean food consumption in grams per animal and day.
Body weight data: The body weight was determined before the administration period in order to animals. During the conduct of the study, the body weight was determined on day 0 (start administration period) and thereafter in weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.
Administration volume: The administration volume was 10 ml/kg body weight. The amounts of test substance preparation to be administered to each animal were calculated once a week on the day of body weight determination.
The animals were examined for evident signs of toxicity and mortality twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and on public holidays). The animals were checked for abnormal clinical signs each time before and after dosing. In order to prepare tables of the clinical symptoms observed, the data were entered manually into the computer systems; according to the particular health status of the individual animals, sometimes several, different clinical signs were documented during one week. As these tables represent summaries of all clinical symptoms observed during one week - including "nothing abnormal detected" - several findings can be found during one observation period.
Urinalyses, as well as selected clinicochemical examinations were carried out in all animals during the last week of dosing.
One male and female of each group was perfused with 4% formaldehyde solution.

Sacrifice and pathology:

All animals were assessed by gross pathology; the kidneys were examined histopathologically (all animals) and by electron microscopy (selected animals, only).

Results and discussion

Results of examinations

Details on results:

1500 mg/kg body weight: impairment in food consumption in males and to a lower extent also in females; impairment in body weight in males and transiently also in females; clinical symptoms in males and females (diarrhea, anogenital region smeared with urine, crust formation at the nose and/or piloerection); increase in serum urea, renal tubular and transitional epithelial cells in both sexes; urinary excretion of crystalline CaNaNTA by isolated animals of both sexes; hematuria in males; increased excretion of renal (granular) casts by females; increase of mean relative kidney weight in males; increase of mean absolute and relative kidney weight in females; cytoplasmic vacuolization of the proximal tubule in 4 males and 3 females; degeneration of the pars recta in 3 males and 1 female; basophilic (regenerating) tubules in an increased severity in males and females; tubular hyperplasia in 2 males; vesiculation and vacuolation of the rough endoplasmatic reticulum of proximal tubule cells, predominantly of the S3 segment.
500 mg/kg body weight: slight impairment in body weight in males; increase of mean relative kidney weight in females; cytoplasmic vacuolization of the proximal tubule in 4 males and 5 females; degeneration of the pars recta in 1 male; basophilic (regenerating) tubules in an increased severity in males and females.
150 mg/kg body weight: no substance-related findings.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

In both sexes of the high dose group, food consumption and body weight were affected and the animals exhibited clinical signs of toxicity (e.g. diarrhea). In the mid dose group still a slight impairment of body weight was seen in males. Changes in clinical pathology testing were seen in the high dose animals only. Most of these findings are indicative of functional impairment of kidney. The occurrence of renal tubular and transitional epithelial cells as well as granular casts in the urine specimens are clear signs of tubular damage or injury. Furthermore, the slightly increased urea concentrations in the sera of the high dose animals and hematuria in the high dose males are also indicative of kidney damage. The lowered urine pH is probably a consequence of the pH effect of increased concentrations of the acidic test compound in the urine specimens and the crystals of "unknown origin" seen in isolated animals of both sexes in the highest dose group presumably consist of crystalline CaNaNTA. Kidney weights were increased in high dose males (relative weights) and females (absolute and relative weights) and in mid dose females (relative weights). This increase in weight correlates to a vacuolization of the proximal tubule, degeneration of the pars recta, and to an increased severity of basophilic (regenerative) tubules.

Applicant's summary and conclusion

Conclusions:

Thus, substance-related signs of nephrotoxicity were seen at dose levels of 1500 and 500 mg/kg body weight. The strain of rats used therefore is sensitive for nephrotoxic changes after repeated administration of NTA.