Trisodium nitrilotriacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996-01-15 to 1997-12-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study; meets generally accepted scientific standards and is described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

- missing hematology examinations, incomplete clinical biochemistry, and incomplete list of organs examined for histopathology

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

- missing hematology examinations, incomplete clinical biochemistry, and incomplete list of organs examined for histopathology

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium nitrilotriacetate

EC Number:

225-768-6

EC Name:

Trisodium nitrilotriacetate

Cas Number:

5064-31-3

Molecular formula:

C6H9NO6.3Na

IUPAC Name:

trisodium 2-[bis(carboxylatomethyl)amino]acetate

Details on test material:

- Name of test material (as cited in study report):Na3NTA (Trilon A92), Tri-sodiumsalt of Nitrolotriacetic acid, Monohydrate
- Physical state: solid white powder
- Analytical purity: 92.4% (IR- and UV-Spectroscopy)
- Lot/batch No.: Abl.No. 05-5056 (Test No.95/61)
- Stability under test conditions: stable - proven by reanalysis after the in-life phase of the study
- Storage condition of test material: room temperature
- Other: date of production: 1995-02-08

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 14 weeks
- Weight at study initiation: 355-408 g (mean 377 g)
- Fasting period before study: none
- Housing: individual in DK III stainless steel wire mesh cages (floor area about 800 cm²)
- Diet (e.g. ad libitum): ad libitum - ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingenthalmühle AG, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days/one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Air changes (per hr): fully air conditioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1996-01-22 To: 1996-02-22

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): mixture of food and test substance was prepared once before the start of the study
- Mixing appropriate amounts with (Type of food): 150 ppm (9 mg/kg bw/day) and 20,000 ppm (926 mg/kg bw/day) was prepared by mixing weight out amount of test substance with small amount of food in a beaker. Subsequently a premix was prepared by adding an appropriate amount of food (Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingenthalmühle AG, Kaiseraugst, Switzerland).
- Storage temperature of food: premixed feeding mixture was frozen, and weekly aliquots thawed, warmed to room temperature and offered to the animals for one week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of Na3NTA preparations was proven at 150 and 20,000 ppm at the start of the administration period

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Prepared weekly mixtures offered to the animals for one week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 20000 ppm (0, 9; 926 mg/kg/d)
Basis:
nominal in diet

No. of animals per sex per dose:

10 (150 ppm)
20 (0 and 20,000 ppm)

Control animals:

yes

Details on study design:

- Dose selection rationale: Previous study administered 20,000 ppm NaNTA over 18-months with 6-months recovery period and determined kidney tumors in rats. No such reaction was noted in a similar study using different species and 300 ppm test substance concentration. The study was set up to have both, effects and no effects, hence 150 ppm and 20,000 ppm were selected.
- Rationale for animal assignment (if not random): grouping by weight but group assignment at random
- Post-exposure recovery period in satellite groups: no recovery period
- Section schedule rationale (if not random): random

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: morning and late afternoon
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week. Results listed in table No.IA-004

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
Results listed in tables No.IA-012 to IA-015

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Results listed in tables No.IA-005-IA-007

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No drinking water study
- Time schedule for examinations: determined weekly over a period of 4 days and calculated as mean drinking water consumption in g per animal per day
Results listed in tables No.IA-008 to IA-011

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Not examined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at the end of the study period (day 30) from the retroorbital venous plexus in the morning
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5
- Parameters Transferin (TRFEERR), iron and total iron-binding capacity (TEBK) were checked. Results are compiled in table No.IB-001.
In addition the following Parameters were determined in the urine obtained: creatine (UCREA), lactate dehydrogenase (ULHD), alkaline phosphatase (UALP), gamma-glutamyltransferase (UGGT), alanine aminopeptidase (UAAP), N-acetyl-ß-glucosaminidase (UNAG), calcium (UCA and UCALC), iron (UFE and UIRON) as well as zinc (UZN and UZINC) . Results listed in table No.IB-002 - IB-005, as well as IB-006 to IB-015.

URINALYSIS: Yes
- Time schedule for collection of urine: collected overnight at about 4°C
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters volume, color, turbidity, nitrate, protein, glucose, ketones, urobilinogen, bilirubin, blood, pH, specific gravity as well as sediment were examined. Results listed in table No.IB-002 - IB-005, as well as IB-006 to IB-015.

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:
Biochemistry: determination of 8-OH-2-deoxyguanosine and lipid peroxidation in kidneys
Macroscopic Pathology and Paraffin Histology of perfused animals
Ultrastructural pathology of perfused animals (the kidneys)
Ultrastructural pathology of non-perfused animals (the kidneys) and determination of DNA-synthesis in the kidneys.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Means and standard deviations were calculated for each test group using the parametric one-way analysis of the F-test (ANOVA, two-sided) and DUNNETT's two-sided test for the hypothesis of equal means was performed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One animal of the 20,000 ppm test group died

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal of the 20,000 ppm test group died

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no substance related effects at 150 ppm, but decreased terminal body weight gain at 20,000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

no substance related effects at 150 ppm, but reduced food consumption at 20,000 ppm (- 47.8%).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Animals of the 20,000 ppm test group showed statistically increased daily consumption (up to 102.4%)

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of the 20,000 ppm Na3NTA test group revealed increased blood and lactate deydrogenase after 2, 4 and 8 days of administration. Further the y-glutamyltransferase and ceratinine concentrations were reduced after 8 days.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
At 20000 ppm Na3NTA group one animal died on day 28, all animals had red-brown discoloured urine from day 9 to the end of the study, some of the animals showed piloerection from day 21 on. No effects at the 150 ppm dose level.

BODY WEIGHT AND WEIGHT GAIN
At 20,000 ppm dose level, food consumption was decreased up to 48% during the entire study and water consumption increased up to 102% of the controls. The body weights were significantly lower (-26%) than in the control groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Based on the daily test substance intake of the test groups and body weight over the entire study period the following was calculated:
150 -> 9 mg/kg bw/day and
20,000 ppm -> 926 mg/kg bw/day

CLINICAL CHEMISTRY
Clinical chemistry examinations did not reveal changes of the levels of iron concentrations, transferrin levels and total iron binding capacity.
Quantitative measurements in the urine revealed increased activities of LDH on day 2, 4 and 8 of the study. At day 8, y-GGT and creatinine concentrations were reduced. Increased concentrations of zinc were excreted with the urine at the end of the study, but calcium and iron excretion remained unaffected.

URINALYSIS
Standard urinalysis revealed macrohematuria on day 8, increased blood and in the sediment elevated number of erythrocytes on day 4, 8, and 29. The mean specific gravity was reduced and the mean urine volume was increased (none significantly) on day 8 and 29. The urine specimens were discoloured from dark yellow to light brown and appeared cloudy on days 2, 4 and 8. Decreased amounts of urinary crystals were found on day 2, 4 and 8 and increased numbers of transitional epithelial were found on day 4 and 8 of the treatment.

ORGAN WEIGHTS: body weight was significantly reduced, while the absolute and relative weights of kidneys were increased in high dose group.

GROSS PATHOLOGY
All animals of the high dose group showed enlarged kidneys and dilation of the renal pelvis. The ureters were dilated in one animal. Microscopic lesions were seen in the kidney of all high dose animals consisting of tubular hyperplasia. These hyperplasias were characterised by tubules with large, vacuolized cells. Other changes in most or all animals of this group were basophilia, vacuolation (without hyperplasia), dilation, and calcification of tubules, interstitial nephritis, inflammation, necrosis, fibrosis and urothelial hyperplasia of the renal papilla and pelvic dilation.

HISTOPATHOLOGY: NEOPLASTIC
The severity of hyperplastic lesions was associated with the occurrence of pelvic dilation and loss/necrosis of the renal papilla. Iron particles of interstitial macrophages positive with Perl’s iron staining (Fe2+ and Fe3+) were similarly distributed between the treated and control groups. No positive reaction was found with Turnbull’s stain (Fe2+) indicating that the observed particles were likely to represent hemosiderin.

OTHER FINDINGS - ULTRASTRUCTURAL PATHOLOGY OF PERFUSED ANIMALS(4 weeks)
Ultra structurally, the histomorphological vacuolation of tubular epithelial cells were confirmed to consist of different stages of vesiculation and dilation of the rough endoplasmic endothelium, occasionally accompanied by cytoplasmic blebbing into the tubular lumen, and of ballooning degeneration of mitochondria. These changes were found in samples of the cortex and outer medulla, but not in the inner medullar regions. They are characteristic for different stages of cell swelling and vacuolar degeneration up to lysis of cells.

ULTRASTRUCTURAL PATHOLOGY OF NON-PERFUSED ANIMALS, DETERMINATION OF DNA-SYSTHESIS AND DETECTION OF FERRITIN
There were no gross lesions detectable after one week of administration. After 4 weeks of treatment, three animals showed dilation of the renal pelvis and the ureters. No lesions related to the Na3NTA treatment were observed in the liver, pancreas, and spleen. One animal died intercurrently.
8-OH-dG levels (measured by HPLC) were not increased after treatment (0.65 8-OH-dG per 105 dG in the negative control group, 0.01 and 0.84 in the treated groups). In the 20000 ppm-group, BrdUrd labelling was used to determine frequencies of S-phase cells in the cortex, the outer stripe of the outer medulla and the inner stripe of the outer medulla. No effect on cell replication was found in the cortex region after exposure for 1 week, whereas 10- to 19-fold increases in S-phase cells were observed after 4-week exposure.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

150 ppm

Sex:

male

Basis for effect level:

other: overall toxicity

2

Dose descriptor:

LOAEL

Effect level:

20 000 ppm

Sex:

male

Basis for effect level:

other: kidney toxicity

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

In this study the effects of Na3NTA were compared with those of FeNTA (i.p.). FeNTA related effets differs in extent, pattern and mechanism of inducing kidney toxicity. The results indicate that Na3NTA related effects are not mediated by an internal formation of FeNTA.

Applicant's summary and conclusion

Conclusions:

The NOAEL of this subacute oral toxicity study was 150 ppm Na3NTA.H2O (9 mg/kg bw/d). Substance related effects at the high dosage (20,000 ppm) included kidney toxicity, reduced food consumption and significant lower body weight gain than control groups.

Executive summary:

In a subacute toxicity study Na3NTA (92.4%) was administered to male Wistar rats at dietary concentrations of 0, 150 and 20000 ppm (appr. 9 and 926 mg/kg bw/d) for 4 weeks.

Subgroups of 5 animals received 150 ppm (2 groups), 20000 ppm (4 groups) or served as controls (4 groups). Clinical examination, urinanalysis, blood examinations, determination of 8-OH-2-dG and of lipid peroxidation in kidneys, histopathology of several organs and ultrastructural pathology of the kidneys have been carried out, as well as determination of DNE-synthesis in the kidney. The results of oral application of were compared to i.p. application of FeNTA. FeNTA related effects differs in extent, pattern and mechanism of inducing kidney toxicity showing that Na3NTA related effects are not mediated by an internal formation of FeNTA.

The NOAEL of this study is 150 ppm Na3NTA.H2O (9 mg/kg bw/d). The LOAEL is 20.000 ppm based on kidney toxicity.

Due to the missing hematology examinations, the incomplete clinical biochemistry, and the incomplete list of organs examined for histopathology, this study did not fulfill the requirements of current standard test protocols of the OECD 407 guideline.