Trisodium nitrilotriacetate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1971

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment (pre-OECD /pre-GLP study)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1971

Report date:

1971

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Repeated exposure of rats to Na3NTA as aerosol.
Examinations: Body weights, clinical observations, Hematology and clinical chemistry, Urine Analysis and respiratory physiology.

GLP compliance:

no

Remarks:

pre-GLP study

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:

- Substance type: pure substance
- Physical state: solid, finely ground powder, pale yellow in colour
- Analytical purity: no data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley albino rats were obtained from Manor Research, Staatsburg, New York.
- Diet (e.g. ad libitum): Animals were fed once a day during the four weeks of exposure after termination of the daily exposure.
- Water (e.g. ad libitum): no data

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Chamber No. mean gravimetric % particles < 10 µm mass median
concentration ± S.E. in diameter ± S.E. diameter ± S.E.
µg/l µg/l % % µm µm
I 0 -- -- -- -- --
II 10.2 1.13 85 0.00 6.0 0.00
III 213.1 15.82 86 3.51 5.5 0.05
IV 342.2 35.15 83 6.52 5.5 0.09

S.E. = standard error

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The chambers were fabricated from 304 L stainless steel with glass windows and are 182,9 cm x 182,9 cm x 182,9 cm with pyramidal tops and bottoms. The test material was introduced into a tube which is tangential to the mixing turret on top of the chamber. The test material is carried along by room air which has passed through a charcoal bed and a Cambridge absolute filter.

- Method of holding animals in test chamber: Each chamber was designed to house a total of nine monkey cages, each holing one animal. The four cages used in this study were randomly distributed throughout each of the nine positions during the course of the exposures. Each cage was made from stainless steel bars and the dimensions are 61 cm high, 42 cm wide and 53 cm long.
Each chamber also held four rodent cages in which the guinea pigs and rats were housed. The sexes of each species were separated from each other. The cages were made from stainless steel wire mesh to allow the maximum exposure of the rodents to airborne test materials. In some instances, the cages were placed in tiers and rotated on a daily basis. In other cases, the cages were placed side by side at the level of the monkey cages and also rotated on a daily basis.

- System of generating particulates/aerosols:
Two methods for the generation of aerosols were employed in this experiment.
When it was desirable to attain atmospheric concentrations of less than 20 micrograms per liter, the Wright Dust Feed was used. This type of dust generator operates by disseminating the powder through the operation of a series of different sized gears which makes it possible to accurately control the quantity of material metered into the chamber.
To attain chamber concentrations from 20 µg/l to 500 µg/l, the pulse-puff aerosol generator was employed. This generator operates by the passage of compressed air through the bottom of the generator which suspends the powder in the generator. Blasts of air at regular intervals insure the suspension of the material which is then forced into the chamber plenum by an additional compressed air source at the top of the generator.
The material is carried along into the mixing turret of the chamber by filtered house air which is continually being passed through the plenum.

- Temperature, humidity, pressure in air chamber: 23,9 °C ± 1°C, humidity: 50% ± 5%
- Air flow rate: 1000 liters/minute
- Method of particle size determination:

- Treatment of exhaust air: The effluent from the chamber was exhausted from the bottom of the chamber and passed through a particulate filter, an activated charcoal filter, and then through a final filter before being passed into the atmosphere.

TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric samples for particle size analysis were obtained with a Monsanto Cascade Impactor and an Andersen Particle Sizing Air Sampler.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Concentration of test material in vehicle: 0.0102, 0.2131, 0.3422 mg/l

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The weekly mean chamber concentrations ± standard error were determined by gravimetric analysis.
The atmospheric concentration of the test materials in the chambers was determined gravimetrically. Samples of the chamber atmosphere were obtained from the breathing zone of the monkeys (see 7.5.3 PUG1971-Na3NTA-4-weeks repeated inhalational exposure-repeated dose toxicity_monkeys) by drawing volumes of air through a sampling tube. The sampled materials were collected by a MSA glass fiber filter which was connected to the sampling tube. These filters were then weighed on an analytical balance. In each case the volume of air which was passed through the filter was recorded. The concentration of the aerosol in the chamber was determined by dividing the total quantity (weight) of powder collected by the volume (liters) of air in that sample.
The atmospheric concentrations were determined routinely by this method. However, to insure the validity of this method, these concentrations were corroborated by employing the Monsanto Cascade Impactor and the Andersen Particle-Sizing Air Sampler (Model II 0705) which are normally used for particle size determinations, but which can be employed also for the quantitation of atmospheric concentrations.
In order to insure that the chamber concentrations remained relatively constant and at the proper concentration during the exposure, a number of samples were obtained during each exposure and immediately analyzed.

Duration of treatment / exposure:

6 hours a day

Frequency of treatment:

5 days a week for 4 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.0102, 0.2131, 0.3422 mg/l
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.0, 0.010, 0.250, 0.500 mg/l
Basis:
nominal conc.

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, beginning prior to the first exposure, during the four weeks exposure and the two weeks post exposure period

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from the abdominal aorta of each rat on day 28 and 42 at the time of sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked in table [No.1] were examined.
- The biochemical determinations were performed on the serum automatically by a Technicon SMA-12 Auto Analyzer

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from the abdominal aorta of each rat on day 28 and 42 at the time of sacrifice
- Animals fasted: No data
- How many animals: all
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to the initial inhalation exposure f of each rat and on day 28 and 42
- Metabolism cages used for collection of urine of rats: Yes
- Animals fasted: During the collection period (early afternoon till next morning), the rats did not receive any food, however,
water was available ad libitum.
- Parameters examined: calcium, copper, iron, magnesium, and zinc. Method: atomic absorption

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes :

brain liver adrenal gland
pituitary gland heart urinary bladder
thyroid gland spleen peribronchial lymph nodes
trachea stomach peripheral nerve
lungs large intestine seminal vesicles and prostate gland
pancreas small intestine uterus
esophagus kidneys skin
salivary gland gonads
eyes muscle

HISTOPATHOLOGY: Yes:
histological preparation of:
lungs spleen
trachea adrenal gland
liver peribronchial lymph nodes
kidneys stomach
small intestine urinary bladder
large intestine gonads
heart



All animals which died during the test period were immediately necropsied. One-half of all surviving animals were sacrificed and necropsied an Day 28; the remaining animals were necropsied an Day 42. At necropsy, the Organs were observed for gross lesions. The following tissues were removed and fixed in 102; neutral buffered formalin.

Other examinations:

none

Statistics:

The appropriate data were submitted to the following statistical analysis:
where a comparison of the difference between means was to be performed, a Chi square test was performed to determine heterogeneity of means in the various groups. If there was significant heterogeneity among means, the Student's "t" test was performed at the 0.05 probability level to evaluate the difference between means.
When there was not significant heterogeneity among means as determined by the Chi square test, analysis of variance, as determined by the F-test, was performed. The "t" test was then performed to determine statistically significant differences between means also at the 0.05 probability level.
At each interval, Pre-exposure, Day 27 (28), and Day 41 (42), the means of each experimental group were compared to the control group for that same interval.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no mortality, some evidence of respiratary distress

Mortality:

mortality observed, treatment-related

Description (incidence):

no mortality, some evidence of respiratary distress

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Rats exposed to Na3NTA at 10.2 and 213.1 µg/l did not exhibit any apparent manifestations of discomfort or irritation during the exposure or post exposure periods.
At 342.2 µg/l of Na3NTA, there was some evidente of respiratary distress exhibited by the rats during the second week of exposure.

BODY WEIGHT AND WEIGHT GAIN:
The growth curves exhibited by male adn female rats appeared normal throughout the four-week exposure period and two-week postexposure
observation period when compared to that of the control group for all exposure concentrations.


FOOD CONSUMPTION: no data

FOOD EFFICIENCY: no data

WATER CONSUMPTION: no data

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY:
Hematological determinations performed on rats following the last exposure (Day 28), and two weeks thereafter (Day 42) did not reveal any abnormalities er trends of effect which could be considered to be compound related.

CLINICAL CHEMISTRY:
Biochemical determinations performed on the blood of rats exposed to NA3NTA resulted in statistically significant increases at the Day 28 interval in serum glutamic oxaloacetic transaminase, albumin, globulin, total protein, and a corresponding increase in the albumin/globulin ratio. There was also an increase in the gamma globulin fraction when electrophoresis was performed on the serum. The control group at this interval did not exhibit elevated values. With the exception of increased gamma-globulin levels, all other values had returned to the control range by Day 42.
It is not be possible to conclude what importance these biochemical findings may have without histopathological examination of the appropriate tissues and correlation of the data. This is not performed within the study.

URINALYSIS:
Analyses of copper, iron, and zinc trace metals in the urine of rats exposed to Na3NTA did not reveal any substantial increases or decreases or trends of effect which could be considered compound related.

NEUROBEHAVIOUR: no data

ORGAN WEIGHTS:
The organ/body weight ratios for rats sacrificed at Days 28 and 42 do not show any anomaly. A careful evaluation of the organ/body weight ratlos did not indicate seemingly abnormal values among any of the animals which were sacrificed at the proper intervals, when compared to the control group.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.213 mg/L air

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

LOAEC

Remarks:

systemic

Effect level:

0.342 mg/L air

Sex:

male/female

Basis for effect level:

other: blood biochemistry: increased total protein, globuin, bilirubin

3

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

0.342 mg/L air

Sex:

male/female

Basis for effect level:

other: sight to severe respiratory distress

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No mortality in either of the animal groups were found. A NOAEC of 0.21 mg/l was established. The LOAEC of 0.34 mg/l based on slight to severe respiratory distress (local) and some changes in blood biochemistry (systemic). All effects disappeared until day 42, the end of the observation period.

Executive summary:

In a 4 -weeks repeated dose inhalation toxicity study rats/sex/concentration were exposed to Na3NTA for 6 h/d (5 d/w) at concentrations of 0, 0.010, 0.213, 0.342 mg/l (non-micronised Na3NTA). Animals then were observed for 14 days.

 

No mortality was observed after treatment with Na3NTA.

No consistent damages were reported.

Some animals of the 0.342 mg/l group (2/12 rats) exhibited dyspnea during the first 2 weeks of exposure. At the same concentation reversible changes in blood biochemisty were reported. 0.342 mg/l is considered the LOAEC.

Necroscopy findings from rats did not reveal any consistent observations which could be attributed to exposure to Na3NTA.