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:

2002-06-11 to 2003-05-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no guideline exists for this type of study - site participates in GLP compliance program; however, no analyses of the test substance in the food were carried out

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

4 week feeding study in male Wistar rats at 0 and 150 ppm, focussing on kidney toxicity.

GLP compliance:

yes (incl. QA statement)

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): Trilon A92 R; Monohydrat des Trinatriumsalzes der Nitrilotriessigsäure
- Physical state: solid white powder
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed by the sponsor and the sponsor holds this responsibility.
- Storage condition of test material: room temperature; test substance is hydroscopic
- manufacturing date: 2002-05-02
- Other: Homogeneity verified

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 13 weeks
- Weight at study initiation:
- Fasting period before study: none
- Housing: individually in DKIII stainless steel wire mesh cages (floor area about 800 cm²). Underneath waste trays containing adsorbent material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 2002-06-18 To: 2002-07-17

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): mixtures were prepared weekly
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet rat/mouse meal, supplied by Provimi Kliba AG, Kaiseraugst, Switzerland

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analyses of the test substance in the diet were carried out

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 males

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Previous study administered 150 ppm Na3NTA over 28-day period and determined the 8-OH-2-deoxyguanosine content in kidney to be significantly lower than in controls (BASF 99S0061/95057). The study was set up to reproduce this effect and test if the substance may have a protective effect on the kidney at low concentrations.
- Rationale for animal assignment (if not random): random

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (morning and late afternoon), once a day on Saturdays, Sundays and public holidays
- Cage side observations checked in table [No.IA-001] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at start of administration period and weekly thereafter

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

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- 8-HO-deoxyguanosine was determined in the right kidney and lipid peroxidation was determined in the left kidney.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: BIOCHEMICAL INVESTIGATION: determination of 8-OH-2-deoxyguanosine content in the right kidney (according to Dahlhaus et al. 1993 and Fialy et al. 1989) and lipid peroxidation in the left kidney (according to Reece et al. 1998). (see table IB-001 - IIA-003)

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Other examinations:

At necropsy, kidneys were weighted and 8-HO-deoxyguanosine was determined in the right kidney and lipid peroxidation was determined in the left kidney.

Statistics:

Mean and standard deviation of each test group were calculated for several parameters including body weight, body weight change, food consumption, water consumption, and food efficiency. A comparison of each group with the control group was performed using DUNNETT’s test (two-sided) for the hypothesis of equal means.

* for p ≤ 0.05
** for p ≤ 0.01

Dunnett C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol.50, 1096 - 1121
Dunnett C.W. (1964): New tables for multiple comparisons with a control. Biometrics, Vol.20, 482 - 491

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Lipid peroxidation and 8-HO-Deoxyguanosine level in kidney DNA

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

only kidney examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: no animal died during the study

BODY WEIGHT AND WEIGHT GAIN: no substance-related effects observed

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no substance-related effects observed

FOOD EFFICIENCY: no substance-related effects noted

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no substance-related effects observed

The following examinations were not performed: OPHTHALMOSCOPIC EXAMINATION, HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS, NEUROBEHAVIOUR, GROSS PATHOLOGY, HISTOPATHOLOGY: NON-NEOPLASTIC, HISTOPATHOLOGY: NEOPLASTIC , and HISTORICAL CONTROL DATA .

ORGAN WEIGHTS determined only on kidneys: no substance-related effects observed

OTHER FINDINGS: determination of the 8-HO-2-deoxyguanosine (8-HO-dG) content noted a statistically significant reduction in the 8-HO-dG level (by 35% in kidney DNA of rats treated with NTA).

Effect levels

Dose descriptor:

LOEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decrease of 8-HO-dG levels in kidney DNA (suggesting protective effect)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

	Group 0: Control	Group 1: 150 ppm NTA
Kidney weight	0.9559	0.9939
Mean TBA-reactive material (MDA-equivalents) [nmol/g tissue]	114.7 (±22.1)	114.3 (±20.9)
Mean 8-OH-dG-level [8 -HO-dG/10^5dG]	131.2 (± 54.0)	85.9 (±38.9)

Applicant's summary and conclusion

Conclusions:

Administering 150 ppm Na3NTA over 28 days to male Wistar rats did not affect kidney weight but caused a significant decrease in the 8-HO-dG-level in kidney DNA. This is considered to be biologically relevant (oxidative stress), and confirms the analysis of a previous study (BASF, 1998 (99S0061/95057)).

Executive summary:

In a subchronic toxicity study Na3NTA (92.4%) was administered to male Wistar rats at dietary concentrations of 150 ppm (appr. 9 mg/kg bw/d) for 4 weeks. There is no guideline for such subchronic toxicity study to assess oxidative stress in kidneys using male Wistar rats.

Subgroups of 10 animals received 0 (control group) and 150 ppm Na3NTA. Animals were examined for clinical effects, food consumption, body weight (change), and pathology of the kidneys. 8-HO-deoxyguanosine levels and lipid peroxidation in kidneys (being measured as the amount of TBA-reactive material) was determined as indicators of oxidative stress. Kidney weights and lipid peroxidation were not altered. The 8-HO-dG-level in the kidney was statistically significantly lower in treated rats than the control group. This is considered to be biologically relevant suggesting a protective effect of Na3NTA with regards to oxidative stress in the kidneys. The data confirmed the analysis of a previous study (BASF, 1998 (99S0061/95057).