L-Aspartic acid, N,N'-1,2-ethanediylbis-, sodium salt (1:3)

Endpoint summary

Description of key information

No human data were identified on trisodium EDDS, and no dermal or inhalation repeated dose laboratory animal data were identified.

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 of rats at 700 mg/kg bw/day and above following repeated dietary administration.

In a GLP study designed to assess toxicity and mineral balance, no adverse effects were observed when trisodium EDDS was given to male rats in the diet for 28 days at up to 400 mg/kg bw/day, considered the study NOAEL. A dose-related and statistically significant increase in the urinary excretion of zinc was observed at all tested doses (50 mg/kg bw/day and above; considered the study LOEL), which was compensated for by a decrease in faecal zinc excretion (measured only in the two highest dose groups) so that the overall total excretion of zinc was unaltered.

In a GLP study, trisodium EDDS showed no evidence of toxicity when fed in the diet to male rats for 14 days at up to 1250 mg/kg bw/day, considered the study NOEL.

In a 14-day dietary study, trisodium EDDS exhibited toxicity at 2500 mg/kg bw/day and above in male and female rats, seen as loss of body weight, reduced food and water consumption, diarrhoea and hunched posture. The study NOAEL is therefore 500 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 1993 to 24 March 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

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., W¿lferstrasse 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

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

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

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.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related deaths or clinical signs of toxicity were evident.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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):

effects observed, treatment-related

Description (incidence and severity):

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.

Food efficiency:

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

no effects observed

Description (incidence and severity):

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

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Critical effects observed:

not specified

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 regulation, 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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 May to 4 June 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

narrow range of doses (as the study was designed to measure effects on mineral balance), limited number of organs examined; very limited clinical biochemistry (only creatinine was analysed)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., W¿lferstrasse 4, CH-4414 Fuellinsdorf, Switzerland
- Age at study initiation: 5 weeks
- Weight at study initiation: about 112 g
- Fasting period before study: no data
- Housing: individually in Makrolon cages on autoclaved softwood bedding
- Diet (e.g. ad libitum): conventional, ad libitum
- Water (e.g. ad libitum): double-distilled, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22.5
- Humidity (%): 45-68
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): mixed with microgranulated feed, water added to aid pelleting and the pellets dried in warm air for 2 days
- Storage temperature of food: room temperature in paper bags

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

100 g samples of the diet frozen at -20°C sent away on dry ice for analysis (results not included in the report)

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, ad libitum in diet

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

59.15 mg/kg bw/day (actual dose received)

Dose / conc.:

176.08 mg/kg bw/day (actual dose received)

Dose / conc.:

340.25 mg/kg bw/day (actual dose received)

Dose / conc.:

469.66 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on a 14-day range-finding study
- Rationale for animal assignment (if not random): random

Positive control:

none

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment, then twice 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: measured twice weekly over a 24-h period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization and on day 21 of treatment
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 28
- Anaesthetic used for blood collection: Yes, under ether
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, total leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: creatinine

URINALYSIS: Yes
- Time schedule for collection of urine: collected over a 24-h period on days 26, 27 and 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, specific gravity, osmolality, colour, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, sediment, creatinine and (at day 28 only) creatine clearance

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: mineral content (calcium, sodium, potassium, magnesium, zinc, phosphorus, iron (Fe3+), manganese and copper) of faeces and urine (on day 25 onwards) and liver, femur and sternum (at necropsy) in 0, 300 and 400 mg/kg bw/day groups determined. Zinc levels also determined in urine, faeces and kidney in all groups.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All major organs and tisses examined for abnormalities
HISTOPATHOLOGY: Yes. Adrenal glands, bone (left femur including distal joint), caecum, colon, rectum, left kidney, liver, duodenum, ileum, jejunum, stomach, urinary bladder.

Other examinations:

Bone marrow cytology slides prepared for possible evaluation.

Statistics:

The Dunnett test, Steel test or the exact Fisher test were applied as appropriate

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were evident during the observation period.

Mortality:

no mortality observed

Description (incidence):

No deaths (apart from one animal in the 400 mg/kg bw/day group who died immediately after blood collection on day 29) that were related to treatment occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no statisically significant changes in body weight or body weight gain, however, there was a dose-related trend towards a reduction in body weight and body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related differences in food consumption were evident.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No treatment-related differences in water consumption were evident.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were evident

Haematological findings:

no effects observed

Description (incidence and severity):

The incidence of changes was similar in both control and treated groups

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Creatinine levels were similar in all groups

Urinalysis findings:

no effects observed

Description (incidence and severity):

The incidences of changes were similar in both control and treated groups

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were evident at necropsy

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related abnormalities were observed

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A dose-related statistically significant increase in the zinc content of urine was evident, accompanied by a decreased output in faeces in the 300 and 400 mg/kg bw/day groups (faecal zinc was not determined in the other two treatment groups). Overall there was no change in the total zinc excretion between control and treated groups. Zinc levels in the serum and kidneys were changed in some treated groups but this was not dose-related. Levels of copper in animals given 300 or 400 mg/kg bw/day increased in the urine, and the urinary content of magnesium increased in the highest dose group alone. Changes in other mineral levels were inconsistent and not dose-related and were considered to be incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

LOEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Conclusions:

In a GLP study, no adverse effects were observed when trisodium EDDS was given to male rats in the diet for 28 days at up to 400 mg/kg bw/day, considered the study NOAEL. A dose-related and statistically significant increase in the urinary excretion of zinc was observed at all tested doses (50 mg/kg bw/day and above; considered the study LOEL), which was compensated for by a decrease in faecal zinc excretion (measured only in the two highest dose groups) so that the overall total excretion of zinc was unaltered.

Executive summary:

In a GLP study, trisodium EDDS was assessed for toxicity and its effect on mineral balance in a 28-day dietary study in Wistar rats. Groups of five male rats were fed a diet containing either 0, 50, 150, 300 or 400 mg/kg bw/day of the test substance for 28 days. The animals were examined daily for clinical signs of toxicity. Urine and faeces were collected over the last 3 days of the study for analysis of mineral content (calcium, sodium, potassium, magnesium, zinc, phosphorus, iron, manganese and copper) and blood was sampled before study termination. Ophthalmoscopic examination was performed pretest and on day 21. At necropsy organs and tissues were examined for gross abnormalities and a limited number of organs were subjected to microscopic examination. Mineral levels were determined in the liver, kidney, right femur and sternum.

There were no treatment-related deaths or signs of clinical toxicity, and body and organ weights and food consumption were similar in treated and control animals. At necropsy, no macroscopic or microscopic abnormalities were evident and haematology, ophthalmoscopic examination and urinalysis showed no evidence of treatment-related changes. The NOAEL was therefore determined as 400 mg/kg bw/day. A dose-related and statistically significant increase in the zinc content of urine was evident at all doses (50 mg/kg bw/day and above; considered the study LOEL). However, this increase was compensated for by a decreased zinc output in faeces in the 300 and 400 mg/kg bw/day groups (faecal zinc was not determined in the other two treatment groups), resulting in no overall change in total zinc excretion. Levels of copper in animals given 300 or 400 mg/kg bw/day increased in the urine, and the urinary content of magnesium increased in the highest dose group alone. Changes in other mineral levels were inconsistent and not dose-related and were considered to be incidental.

In conclusion, a 28-d LOEL and NOAEL of 50 and 400 mg/kg bw/day, respectively, were established for trisodium EDDS.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 October to 1 November 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

Dietary study in groups of 5 male rats at three dose levels and a control group in which the animals were observed over a 14-day period for signs of toxicity and subjected to macroscopic examination at necropsy

Only one sex studied; no haematology, clinical chemistry, urinalysis or histopatholgy performed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Biological Research Laboratories Ltd., CH 4414 F¿llinsdorf, Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: mean about 155 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: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 46-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): mixed with microgranulated feed, then water added to aid pelleting and the pellets dried in warm air for 2 days before storage
- Storage temperature of food: at room temperature in paper bags

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily, ad libitum in feed

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Dose / conc.:

691 mg/kg bw/day (actual dose received)

Dose / conc.:

892.2 mg/kg bw/day (actual dose received)

Dose / conc.:

1 204.6 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: to indicate possible target organs for a subsequent 90-day study
- Rationale for animal assignment (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes (particularly checking for signs of diarrhoea)
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: before start of study, then twice 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: no


HAEMATOLOGY: no


CLINICAL CHEMISTRY: no


URINALYSIS: no


NEUROBEHAVIOURAL EXAMINATION: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. Samples of left femur, caecum, colon, jejunum, kidneys, liver, rectum, stomach, and any gross lesions

Other examinations:

none

Statistics:

Dunnett's test was applied to compare differences between the treated and control groups.

Clinical signs:

no effects observed

Description (incidence and severity):

No other clinical signs of toxicity were evident

Mortality:

no mortality observed

Description (incidence):

No deaths occurred during the study

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and weight gain were similar in the treated and control groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences between groups were evident in food consumption and compound intake

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related adverse effects were observed on macroscopic examination in any animal

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

No microscopic findings were reported

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Conclusions:

In a GLP study, trisodium EDDS showed no evidence of toxicity when fed in the diet to male rats for 14 days at up to 1250 mg/kg bw/day, considered the study NOEL.

Executive summary:

In a GLP study, trisodium EDDS was assessed for its toxicity in a 14-day dietary study in male Wistar rats. Groups of 5 animals per dose were fed the test substance at 0, 750, 1000 or 1250 mg/kg bw/day in pelleted diet and observed for clinical signs of toxicity, food consumption, body weight gain and at study termination the internal organs were macroscopically examined for evidence of adverse effects.

No animals died during the study and no differences were seen in body weight gain or food consumption between the groups. No clinical signs of toxicity were observed during the study and on necropsy no macroscopic findings were evident.

In conclusion, in a 14-day dietary study, trisodium EDDS showed no evidence of toxicity when fed to male rats at up to 1250 mg/kg bw/day, considered the no-observed-effect level (NOEL) in this study.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 March to 4 April 1993

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Groups of rats of each sex were fed the test compound at levels of up to 5000 mg/kg bw/day for 14 days. The animals were observed for clinical signs of toxicity, food and water consumption and changes in body weight gain. At necropsy the organs were examined macroscopically.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: males, about 170 g; females, about 108 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily, ad libitum in feed

Remarks:

Doses / Concentrations:
0, 50, 500, 2500 or 5000 mg/kg bw/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 35.35-49.05, 362.26-548.73, 502.82-1910.43, 777.09-2964.88 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: range-finding study to indicate potential target organs and to determine dose levels for subsequent studies
- Rationale for animal assignment (if not random): random

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: days 1,5, 11 and 14


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: No


HAEMATOLOGY: No


CLINICAL CHEMISTRY: No


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, extent of examination unclear (but included the kidneys)

Other examinations:

none

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male in the high-dose group died on day 9 of treatment and the other rats, both male and female, in this group were killed in extremis on day 10. Ruffled fur, diarrhoea, emaciation and hunched posture were noted in both sexes treated at 2500 or 5000 mg/kg bw/day. Sedation was also observed in the highest dose group. No deaths seen at up to 2500 mg/kg bw/day and no clinical signs at up to 500 mg/kg bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male in the high-dose group died on day 9 of treatment and the other rats, both male and female, in this group were killed in extremis on day 10. Ruffled fur, diarrhoea, emaciation and hunched posture were noted in both sexes treated at 2500 or 5000 mg/kg bw/day. Sedation was also observed in the highest dose group. No deaths seen at up to 2500 mg/kg bw/day and no clinical signs at up to 500 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the top dose, body weight loss was evident between days 1 and 8 of treatment. At 2500 mg/kg bw/day a reduction in body weight gain was noted between days 1-5 followed by body weight loss for the rest of the study. No effects on body weight were seen at up to 500 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food consumption was reduced during the entire period of treatment in groups fed 2500 mg/kg bw/day or during the first week in those receiving the top dose. In females, reduced food consumption occurred during the first three days of treatment in all groups, after which it remained reduced at the two highest dose levels only.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was reduced for both sexes in the high-dose group from day 6-10 and at 2500 mg/kg bw/day it was reduced during the second week in males and on days 13-14 in females.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test compound-related changes in organ weights were evident.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related abnomalities were seen on macroscopic examination.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic findings reported [extent of examination unclear]

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

water consumption and compound intake

Critical effects observed:

not specified

Conclusions:

In a 14-day dietary study, trisodium EDDS exhibited toxicity at 2500 mg/kg bw/day and above in male and female rats, seen as loss of body weight, reduced food and water consumption, diarrhoea and hunched posture. The study NOAEL is therefore 500 mg/kg bw/day.

Executive summary:

Trisodium EDDS was assessed for its toxicity in a 14-day dietary study in male and female Wistar rats. Groups of 5 animals of each sex were fed the test substance at 0, 50, 500, 2500 or 5000 mg/kg bw/day in the diet and observed for clinical signs of toxicity, food and water consumption, body weight gain and at study termination the internal organs were macroscopically examined for evidence of adverse effects. One male in the high-dose died during the study and all other animals at this dose level were killed in extremis on day 10 of the study. Ruffled fur, diarrhoea, emaciation and hunched posture were noted in both males and females treated at 2500 and 5000 mg/kg bw/day, and sedation was also observed in the highest dose group. Food and water consumption were reduced and body weight loss occurred at the two highest doses. No test substance-related changes were noted on macroscopic or microscopic examination.

 

In conclusion, trisodium EDDS exhibited toxicity at 2500 mg/kg bw/day and above, seen as loss of body weight, reduced food and water consumption, diarrhoea and hunched posture, in rats of each sex in a 14-day dietary study. The study no-observed-adverse-effect level (NOAEL) is therefore 500 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 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 assessed. 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, the latter of which 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 (Dotti et al. 1995).

In a GLP study, trisodium EDDS was assessed for toxicity and its effect on mineral balance in a 28-day dietary study in Wistar rats. Groups of five male rats were fed a diet containing 0, 50, 150, 300 or 400 mg/kg bw/day of the test substance for 28 days. The animals were examined daily for clinical signs of toxicity. Urine and faeces were collected over the last 3 days of the study for analysis of mineral content (calcium, sodium, potassium, magnesium, zinc, phosphorus, iron, manganese and copper) and blood was sampled before study termination. Ophthalmoscopic examination was performed pretest and on day 21. At necropsy organs and tissues were examined for gross abnormalities and a limited number of organs were subjected to microscopic examination. Mineral levels were determined in the liver, kidney, right femur and sternum. There were no treatment-related deaths or signs of clinical toxicity, and body and organ weights and food consumption were similar in treated and control animals. At necropsy, no macroscopic or microscopic abnormalities were evident and haematology, ophthalmoscopic examination and urinalysis showed no evidence of treatment-related changes. The NOAEL was therefore determined as 400 mg/kg bw/day. A dose-related and statistically significant increase in the zinc content of urine was evident at all doses (50 mg/kg bw/day and above; considered the study lowest-observed-effect level (LOEL)). However, this increase was compensated for by a decreased zinc output in faeces in the 300 and 400 mg/kg bw/day groups (faecal zinc was not determined in the other two treatment groups), resulting in no overall change in total zinc excretion. Levels of copper in animals given 300 or 400 mg/kg bw/day increased in the urine, and the urinary content of magnesium increased in the highest dose group alone. Changes in other mineral levels were inconsistent and not dose-related and were considered to be incidental. In conclusion, a 28-d LOEL and NOAEL of 50 and 400 mg/kg bw/day, respectively, were established for trisodium EDDS (Dotti, 1995).

In a GLP study, trisodium EDDS was assessed for its toxicity in a 14-day dietary study in male Wistar rats. Groups of 5 animals per dose were fed the test substance at 0, 750, 1000 or 1250 mg/kg bw/day in pelleted diet and observed for clinical signs of toxicity, food consumption, body weight gain and at study termination the internal organs were macroscopically examined for evidence of adverse effects. No animals died during the study and no differences were seen in body weight gain or food consumption between the groups. No clinical signs of toxicity were observed during the study and on necropsy no macroscopic findings were evident. In conclusion, trisodium EDDS showed no evidence of toxicity when fed to male rats at up to 1250 mg/kg bw/day, considered the no-observed-effect level (NOEL) in this study (Dotti and Wilson, 1996).

Trisodium EDDS was assessed for its toxicity in a 14-day dietary study in male and female rats. Groups of 5 animals of each sex were fed the test substance at 0, 50, 500, 2500 or 5000 mg/kg bw/day in the diet and observed for clinical signs of toxicity, food and water consumption, body weight gain and at study termination the internal organs were macroscopically examined for evidence of adverse effects. One male in the high-dose died during the study and all other animals at this dose level were killed in extremis on day 10 of the study. Ruffled fur, diarrhoea, emaciation and hunched posture were noted in both males and females treated at 2500 and 5000 mg/kg bw/day, and sedation was also observed in the highest dose group. Food and water consumption were reduced and body weight loss occurred at the two highest doses. No test substance-related changes were noted on macroscopic or microscopic examination. In conclusion, trisodium EDDS exhibited toxicity at 2500 mg/kg bw/day and above, seen as loss of body weight, reduced food and water consumption, diarrhoea and hunched posture, in rats of each sex in a 14-day dietary study. The study NOAEL is therefore 500 mg/kg bw/day (Dotti, 1993).

No human data were identified on trisodium EDDS, and no dermal or inhalation repeated dose laboratory animal data.

Justification for classification or non-classification

According to EU CLP regulation, 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.