L-Aspartic acid, N,N'-1,2-ethanediylbis-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

23 November 1993 to 24 March 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (with certain deviations form the current version), to GLP; on related material

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

neurological endpoints were not assessed as the study was conducted before the current guideline was published

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

neurological endpoints were not assessed as the study was conducted before the current guideline was published

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium EDDS

IUPAC Name:

Trisodium EDDS

Constituent 2

Reference substance name:

l-aspartic acid, N,N'-1,2-ethanediylbis-, trisodium salt

IUPAC Name:

l-aspartic acid, N,N'-1,2-ethanediylbis-, trisodium salt

Constituent 3

Reference substance name:

178949-82-1

EC Number:

605-842-8

Cas Number:

178949-82-1

IUPAC Name:

178949-82-1

Constituent 4

Reference substance name:

-

EC Number:

416-530-4

EC Name:

-

IUPAC Name:

416-530-4

Details on test material:

- Name of test material (as cited in study report): trisodium EDDS (under code)
- Molecular formula (if other than submission substance): C10-H13-N2-O8. 3Na
- Molecular weight (if other than submission substance): 358
- Smiles notation (if other than submission substance): [Na+].[Na+].[Na+].OC(=O)[C@H](CC([O-])=O)NCCNC(CC([O-])=O)C([O-])=O
- Substance type: technical product
- Physical state: clear liquid
- Purity test date: no data
- Lot/batch No.: 01
- Expiration date of the lot/batch: 1 January 1998
- Stability under test conditions: stable for at least 30 days
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fuellinsdorf, Switzerland
- Age at study initiation: 5 weeks
- Weight at study initiation: males, mean about 120 g; females, mean about 100 g
- Fasting period before study: no data
- Housing: individually in Makrolon cages on softwood bedding
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 44-61
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly until 21 January 1994, then every 2 weeks
- Mixing appropriate amounts with (Type of food): mixed with microgranulated food, then pellets prepared by adding water to aid pelleting and drying for about 2 days before storage.
- Storage temperature of food: room temperature in paper bags

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no details

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, ad libitum in diet

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 300, 700 or 1000 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

20/sex/dose at 0 and 1000 mg/kg bw/day (10/sex/dose kept for a 4-week recovery period)
10/sex/dose at 50, 300 and 700 mg/kg bw/day

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of a 14-day range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: control and highest dose groups selected
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: at start of study, then weekly and at study termination


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: No data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of study and at weeks 4, 13 and 17
- Dose groups that were examined: all groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 4 and 13 weeks (17 weeks for animals in satellite groups)
- Anaesthetic used for blood collection: yes, either at 4 weeks, sodium pentabarbitone at study termination
- Animals fasted: Yes
- How many animals: all


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4 and 13 weeks (17 weeks for animals in satellite groups)
- Animals fasted: Yes
- How many animals: all


URINALYSIS: Yes
- Time schedule for collection of urine: 13 or 17 weeks
- Metabolism cages used for collection of urine: yes
- Animals fasted: urine sampled during the fasting period


NEUROBEHAVIOURAL EXAMINATION: No


OTHER: determination of magnesium, zinc, iron, manganese and copper in plasma samples at week 13 from 10 animals per sex in the control and highest dose groups.
Estrous cycles were determined in 5 females/group in the last 4 weeks of treatment.

Sacrifice and pathology:

GROSS PATHOLOGY: yes, examined for any gross lesions.
HISTOPATHOLOGY: yes. Any gross lesions observed and testes and pancreas in all groups and the following tissues and organs from the control and 1000 mg/kg bw/day groups (including recovery animals):
aorta, bone and bone marrow (sternum and femur), brain, caecum, colon, duodenum, epididymides, esophagus, eyes (with optic nerve and Harderian gland, femur (including joint), heart, ileum, jejunum, kidneys, lacrimal glands exorbital, liver, lungs (infused with formalin), lymph nodes (mandibular, mesenteric), mammary gland, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary gland (mandibular, sublingual), seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland with parathyroid gland, trachea, urinary bladder (infused with formalin), uterus, vagina.

Other examinations:

Sperm analysis in 5 males/dose at end of treatment period, and all males in the control and high dose groups at the end of the recovery period.

Statistics:

The statistical methods of Dunnett, Miller and Fisher were applied as appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related deaths or clinical signs of toxicity were evident.

BODY WEIGHT AND WEIGHT GAIN
At the high-dose, body weight gain was decreased for both sexes, but during the recovery period it increased due to an increase in food consumption (considered a compensatory effect). At 700 mg/kg bw/day there was a slight decrease in body weight gain in males during the last three weeks of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption in most of the treated groups was reduced during the first week of treatment, which was considered to be due to the unpalatability of the test substance. During the second half of the study males in the high-dose group had reduced food consumption which was considered to be treatment-related.

HAEMATOLOGY
A few minor, but statisitically significant, changes occurred in the treated groups. At the high-dose, a slight decrease in haemoglobin and haemoglobin concentration indices were evident in females at 4 weeks and in both sexes at 13 weeks, a slight decrease in haematocrit in males after 4 weeks, and a slight increase in platelets in females at 13 weeks. Slightly reduced thromboplastin time was seen in females in the 300 and 1000 mg/kg bw/day groups at 4 weeks and a slightly prolonged thromboplastin time in males at 300 mg/kg bw/day and above after 13 weeks. These parameters were comparable to the controls at the end of the recovery period.

CLINICAL CHEMISTRY
The slight changes noted were suggestive of metabolic adaptation to the treatment and not considered of toxicological relevance.

URINALYSIS
No treatment-related significant differences were observed between the treated and control groups.

ORGAN WEIGHTS
Differences in organ weights between the groups were not considered to be treatment-related.

GROSS PATHOLOGY
There were no test substance related abnormal findings at macroscopic examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
Pancreas: a dose-related increase in the incidence and severity (slight to moderate) of single cell death in the pancreas was evident (13/20 and 19/20 animals at 700 and 1000 mg/kg bw/day, respectively) but which had regressed by the end of the recovery period. In addition, fatty infiltration which was present in 2/20 and 7/20 animals at 700 and 1000 mg/kg bw/day, respectively, at termination of treatment and was evident in 14/20 animals after the recovery period in the top dose group.

Testes: atypical residual bodies of minimal severity and incidence were noted in the high-dose group (5/20) but these were not apparent following the 4-week recovery period.

OTHER FINDINGS
MINERAL ELEMENTS IN PLASMA
At week 13 in the high-dose groups, there was a significant decrease in plasma zinc levels in male and female animals and plasma copper and magnesium levels in males compared to controls; these effects were considered to be treatment-related.

ESTRUS CYCLE:
There were no adverse effects on the duration of the estrus cycle for any group.

SPERM ANALYSIS:
There were no adverse effects on sperm concentration or motility. In the high-dose group only, there was a significant decrease in normal sperm morphology with a corresponding increase in head-only sperm. Following the recovery period, sperm morphology returned to control levels in all but one animal. The changes in sperm morphology are considered to be related to reduced zinc availability due to the chelating action of the substance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight gain; histopathology (single cell death and fatty infiltration in the pancreas) at 700 and 1000 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a GLP study conducted according to OECD Guideline 408 (available at the time), a 90-d NOAEL of 300 mg/kg bw/day was established for trisodium EDDS based on a reduction in body weight gain and single cell death and fatty infiltration in the pancreas seen at at 700 mg/kg bw/day and above following repeated dietary administration.

Executive summary:

In a GLP study conducted according to OECD Guideline 408 (available at the time), trisodium EDDS was assessed for its ability to cause adverse effects in a 90-day dietary study in male and female Wistar rats.

Groups of 10 animals of each sex were given either 0, 50, 300, 700 or 1000 mg/kg bw/day in a pelleted diet; further groups of 10 animals of each sex were given the control or high-dose diets for 90 days, followed by a 28-day recovery period. Animals were observed for clinical signs of toxicity throughout the study and blood samples were analysed at 4 and 13 weeks (or 17 weeks for the satellite groups). Ophthalmoscopic examinations took place at 4, 13 and 17 weeks. The duration of the estrus cycle was determined in females over the last month of the study. Sperm concentration, motility and morphology in males were also examined. At study termination, animals were examined both macroscopically and microscopically, and absolute and relative organ weights determined.

No treatment-related deaths or clinical signs of toxicity occurred and there were no treatment-related differences in ophthalmoscopic data, organ weights, urinary analysis, clinical chemistry or length of estrus cycle. At 1000 mg/kg bw/day, body weight gain was reduced (as was food consumption in this group) and there were some slight changes in haematological parameters for both sexes (which were no longer statistically significant at the end of the recovery period). At 700 mg/kg bw/day, there was a slight decrease in body weight gain in males during the last three weeks of treatment.

In males at the top dose, an increase in the numbers of abnormal sperm (but no effects on motility or concentration), atypical residual bodies of minimal severity and incidence in the testes, and minimal changes occurred in some clinical biochemical parameters, all of which had mainly regressed by the end of the recovery period. In the pancreas, there was an increased incidence of single cell death and fatty infiltration at 700 mg/kg bw/day and above, and fatty infiltration was still present in the top dose group at the end of the recovery period. At week 13 in the high-dose groups, there was a significant decrease in plasma zinc levels in male and female animals and plasma copper and magnesium levels in males compared to controls; these effects were considered to be treatment-related.

In conclusion, in a 90-day dietary study a no-observed-adverse-effect level (NOAEL) of 300 mg/kg bw/day was established for trisodium EDDS based on a reduction in body weight gain and single cell death and fatty infiltration in the pancreas at 700 mg/kg bw/day and above. According to EU CLP and DSD regulations, trisodium EDDS would not be classified as a specific target organ toxicant under the conditions of this test as the observed critical effects (on the pancreas) were seen at doses significantly higher than 100 mg/kg bw/day.

[Data on trisodium EDDS is considered relevant to use for understanding the potential repeated dose toxicity of EDDS acid, and is acceptable for using as read-across information.]