Disodium 2-hydroxyethyliminodi(acetate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Nov. 21, 1985 to April. 27, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed method comparable to guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-hydroxyethyliminodi(acetate)

EC Number:

205-187-4

EC Name:

Disodium 2-hydroxyethyliminodi(acetate)

Cas Number:

135-37-5

Molecular formula:

C6H9NO5.Na2

IUPAC Name:

disodium 2-hydroxyethyliminodi(acetate)

Constituent 2

Reference substance name:

2-hydroxyethyliminodi(acetic acid)

EC Number:

202-263-9

EC Name:

2-hydroxyethyliminodi(acetic acid)

Cas Number:

93-62-9

Molecular formula:

C6H11NO5

IUPAC Name:

2,2'-[(2-hydroxyethyl)imino]diacetic acid

Details on test material:

- Name of test material: 2-hydroxyethyliminodi(acetic acid) 
-TSIN: E-2775.02 (HEIDA (Hydroxyethyliminodi acetic acid) ex Degussa, 41.1% active),
E-2775.03 (HEIDA ex Degussa Na2 Sol, 50.48% active)
- Substance type: Pure active substance
- Physical state: Colorless liquid 
- Stability under test conditions: Not reported
- Storage condition of test material: The test substance was stored in the dark at room temperature. Formulated test substance was stored at about 4⁰C in a refrigerator.
- Solubility: The test substance was soluble in water
- Other: pH (at 50%) =10.5

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: From Charles River (UK) Ltd., Margate
- Age at study initiation: Approx. 54 days
- Weight at study initiation: 224-317.7 g (males); 134.1-209.2 g (females)
- Fasting period before study: No
- Housing: The animals were housed individually in stainless steel mesh cages suspended over cardboard-lined trays. The cardboard was replaced as often as necessary to maintain hygiene.
- Diet: SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Ground Fine (Special Diets Services Ltd.,Witham), ad libitum, except for an overnight fast before necropsy and blood collection and during urine collection 
- Water: ad libitum, from glass water bottles attached to the cages, the contents were changed daily. The diet and water were considered not to have contained any contaminant at a level which might have affected the integrity or outcome of the study.
- Acclimation period: 26 days. During acclimation, the health status of animals was reassessed and their suitability for experimental purposes confirmed.

ENVIRONMENTAL CONDITIONS 
- Temperature: 19-25 °C
- Humidity: 40-70% 
- Air changes: 15 air changes/h
- Photoperiod: Fluorescent lighting was controlled automatically to give 12 h light/12 h dark cycle/day.

IN-LIFE DATES: From: Nov. 21, 1985 To: April 27, 1986

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

(filtered tap water)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance were prepared daily for 4 weeks and then weekly thereafter. Lot E2775.02 was used for Weeks 1-12 and lot E2775.03 was used in Week 13 only. The latter batch of test substance was partially crystallized at room temperature, unlike the original batch. E2775.03 was warmed to 60⁰C in a water bath prior to formulation as suggested by the sponsor.

- The concentration of the test substance was adjusted weekly on the basis of the predicted mid-week group mean body weight and an estimate of the water consumption.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance formulations for the analysis of achieved concentration were collected in Weeks 1, 5, 8, and 13. The analyses were carried out by the sponsor. The samples were found to be acceptable.

Duration of treatment / exposure:

28 and 91 days

Frequency of treatment:

Continuously in drinking water

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 10, 100 and 500 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

For 28 day (4 week ) duration: 5 animals/sex/dose
For 91 day (13 week) duration: 10 animals/sex/dose
For 91 day + 35 days post observation (treatment free) (19 week) duration: 10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were provided by the sponsor. 

- Rationale for animal assignment: The animals were assigned to treatment groups on arrival using a total randomization procedure. Each animal was permanently numbered (animal identification number) by ear tattoo/notch. Group mean body weights were calculated prior to the start of treatment to ensure no unacceptable differences among groups. Cage positions on the batteries were assigned using a set of random letter permutations.

- Assignment of animals: The animals were assigned into following experimental groups in the study for 28 day (4 week), 91 day (13 week) and 91 day + 35 days post observation (treatment free) (19 week) each :
Group 1(control): Vehicle control
Group 2 (Low dose): 10 mg/kg/day
Group 3 (mid dose): 100 mg/kg/day
Group 4 (High dose): 500 mg/kg/day

Examinations

Observations and examinations performed and frequency:

Mortality/Morbidity: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes  
- Time schedule: Once daily
- Cage side observations included: All animals were examined once daily for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Atweekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on the first day of treatment, at weekly intervals thereafter and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal was determined weekly: Yes
- Compound intake was reported as mg/kg/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes  
- Time schedule for examinations: The weight of water consumed by each animal was determined daily.

OPHTHALMOSCOPIC EXAMINATION: Yes, using a Keeler direct ophthalmoscope with 1% tropicamide as mydriatic agent (instilled into the eyes approx. 15 minutes before the examination).
- Time schedule for examinations: The eyes of all animals were examined before study initiation and during Weeks 4 and 13.  
- Dose groups that were examined: All dose groups

HAEMATOLOGY: Yes  
- Time schedule for collection of blood: 4, 13 and 19 weeks, Blood samples were collected by orbital sinus puncture into tubes containing EDTA anticoagulant for haematology.
- Anaesthetic used for blood collection: Yes under light ether anaesthesia
- Animals fasted: Yes, overnight (about 18 h); water was provided ad libitum.
- How many animals: Samples were collected from all animals designated for the 4 week interim kill in Week 4, from all surviving animals in Week 13, and from the 5 male and 5 female animals from each group with the highest identification numbers surviving to the end of the treatment-free period in Week 19.
- Parameters examined: Haemoglobin concentration, mean cell volume, red cell count and indices (mean cell haemoglobin, packed cell volume, mean cell haemoglobin concentration), total and differential white blood cell count, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4, 13 and 19 weeks, Blood samples were collected by orbital sinus puncture into tubes containing lithium heparin anticoagulant for clinical chemistry.
- Animals fasted: Yes, overnight (about 18 h); water was provided ad libitum.
- How many animals: Samples were collected from all animals designated for the 4 week interim kill in Week 4, from all surviving animals in Week 13, and from the 5 male and 5 female animals from each group with the highest identification numbers surviving to the end of the treatment free period in Week 19. 
- Parameters examined: Alkaline phophatase, glutamate oxaloaxetate transaminase, glutamate pyruvate transaminase, glutamyl transpeptidase, albumin, blood urea nitrogen, creatinine, inorganic phosphorus, sodium, total protein, albumin/globulin ratio, calcium, chloride, potassium, total bilirubin.

URINALYSIS: Yes
- Time schedule for collection of urine: Samples were collected from all animals designated for the Week 4 interim kill in Week 4. In addition samples were taken from the 10 animals of each group and sex with the highest identification numbers in Weeks 4 and 13. Samples were also collected at the end of the treatment free period in Week 19 from the 5 animals of each group and sex with the highest identification numbers. 
- Metabolism cages used for collection of urine: Not reported
- Animals fasted: Yes, overnight
- Parameters examined: Total volume, pH, calcium, iron, manganese, potassium, zinc, specific gravity, urine creatinine, copper, magnesium, phosphorus, sodium, microscopy of spin deposits

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: All designated animals were euthanized by an intraperitoneal injection of sodium pentobarbitone and exsanguinated after 5 (interim sacrifice), 14 (terminal sacrifice) and 19 weeks (sacrifice after treatment free period) of the study. All animals were necropsied in random order and a veterinary pathologist was present at each scheduled necropsy. A full external and internal examination was made with a suitably qualified pathologist in attendance and all gross lesions were recorded.

GROSS PATHOLOGY: Yes, a full external and internal examination was made and all gross lesions were recorded. The following organs were dissected free from fat and contiguous tissue and weighed before fixation: adrenals, kidneys, ovaries, testes, brain, liver and spleen. Paired organs were weighed separately.  

HISTOPATHOLOGY: Yes
- Histopathological examination was performed on following organ/tissues, preserved in 10% neutral buffered formalin (with the exception of the eyes which were fixed in Davidson’s fluid and the bone marrow smears which were fixed in methanol): adrenals, bone marrow smear, caecum, duodenum, eyes and optic nerves, heart, aorta, brain, colon, epididymides, femur (including articular surface), ileum, jejunum, liver, lymph nodes, ovaries, pituitary, rectum, seminal vesicles, skeletal muscle (quadriceps/psoas), spinal cord, sternum (and bone marrow), testes, thyroids (with parathyroids), trachea, uterus, vagina, kidneys, lungs (with mainstem bronchi), oesophagus, pancreas, prostate, salivary gland (submaxillary), sciatic nerve, skin and mammary gland, spleen, stomach, thymus, tongue, urinary bladder, ureter, all gross lesions.

-Initially all tissues from control and high dose groups and all gross lesions from animals sacrificed at Week 4 were embedded in paraffin wax B.P. (m.p. 56 ⁰C), sectioned at a nominal thickness of 5 µm, stained with haematoxylin and eosin and examined by the study pathologist. Sagittal and transverse sections of the femur were prepared.

-Kidneys from all animals in control and high dose groups and all gross lesions from all animals sacrificed in Weeks 13 and 18 were similarly prepared and examined. In addition, the kidneys from group 3 male animals were also examined by the study pathologist.

Statistics:

Data were processed, where appropriate, to give group mean values and standard deviations.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
- One high dose male animal died of multifocal chronic myocarditis during Week 9 and one high dose male died during the treatment free period. Renal tubular vacuolation and basophilia with moderate multifocal cortical mineralization was considered to be the cause of death of the latter animal. There were no treatment-related changes in the clinical condition of the animals.
 
BODY WEIGHT AND WEIGHT GAIN
- There were no treatment-related effects on body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE
- There were no treatment-related effects on food consumption

WATER CONSUMPTION AND COMPOUND INTAKE
- There were no treatment-related effects on water consumption.

OPHTHALMOSCOPIC EXAMINATION
- No test substance-related ophthalmic observations were noted at any dose level tested.

HAEMATOLOGY
- There were no treatment-related effects.

CLINICAL CHEMISTRY
-There were no treatment-related effects.

URINALYSIS
-There was a dose-related increase in the amount of zinc detected in the urine of both male and female treated animals at the Week 4 and 13 analyses. The group mean increases in the high dose group for both sexes were about 250 % in Week 4 and 550-650 % in Week 13, above control values.
-In addition, increases above control levels of copper up to about 200 % were noted in high dose group at both the Week 4 and 13 urine analysis. Levels of urinary iron were also increased above control levels in high dose females by about 380 % at the Week 4 analyses.
- Although the individual values were variable there were slight effects on zinc and copper levels still apparent after the treatment-free period. The increased amounts of zinc, iron and copper ions in the urine of the treated animals may be related to the histopathological lesions in kidney.

ORGAN WEIGHTS
- There were slight increases in male mean relative kidney weights in high dose group animals. These increases occurred in occasional male animals and were up to 17% at the Week 4 necropsies and up to 40 % at the Week 13 necropsies, when compared to respective controls. This increase in relative kidney weights of high dose group males may be related to histopathological lesions in kidney. By the end of the treatment free period this effect was not apparent.
-There were no effects on the organ weights of the female animals that were related to treatment.

GROSS PATHOLOGY
- There were no treatment related macroscopic findings in the Week 4 interim kill or after the 4 week treatment free period. At the Week 13 terminal necropsy 3 males from the high dose group had diminished seminal vesicles and 1 male had enlarged kidneys.

HISTOPATHOLOGY: NON-NEOPLASTIC
-Microscopically, renal tubular vacuolation with a slight increase in the incidence of basophilic tubules was noted in 5 males from high dose group after 13 weeks of treatment. With the exception of one high dose male, which died during the treatment free period and had marked tubular vacuolation of the kidneys, no microscopic observations related to treatment were noted after the 4 week interim sacrifice. Further, the minimal to moderate vacuolation appeared reversible as it was not apparent after 4 weeks without treatment.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Urinalysis, histopathology and gross pathology

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: See above

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of 2 -hydroxyethyliminodiacetic acid to rats at dose levels of 0, 10, 100 and 500 mg test material/kg bw/day in drinking water up to 13 weeks revealed a no observed effect level (NOEL) of 100 mg/kg bw/day (nominal) and of 41 mg/kg bw/day (actual) based on reversible pathological lesions of kidney observed in high dose group animals).

Executive summary:

The sub-chronic toxicity study of 2 -hydroxyethyliminodiacetic acid was performed following the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

The study was designed to evaluate the repeated dose toxicity of the test substance, when administered to Crl:CD(SD)IGS BR rats at dose levels of 0, 10, 100 and 500 mg /kg bw/day in drinking water for a period of 4 and 13 weeks. Additional animals were used to observe any treatment related changes for 35 days after 13 week treatment.

A total of 200 rats of 54 days age (source: Charles River (UK) Ltd., Margate) were used in the study. The body weight range of animals was 224-317.7 g (males); 134.1-209.2 g (females). Animals were housed individually in stainless steel cages suspended over cardboard-lined trays and were maintained under standard laboratory conditions (temperature: 11-26⁰C, humidity: 40-70%, 12 h light/12 h dark cycle per d). Animals were acclimated to laboratory conditions for a period of 26 days and were fed on SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Ground Fine (Special Diets Services Ltd., Witham), ad libitum and received water, ad libitum. There were no known contaminants in the food or water that would have interfered with the study.

The concentration of the test substance was adjusted weekly on the basis of the predicted mid-week group mean body weight and an estimate of the water consumption. Animals were assigned into following experimental groups in the study for 28 days (4 weeks; 5 animals/sex/dose), 91 days (13 weeks; 10 animals/sex/dose) and 91 days + 35 days post observation (treatment free) (19 weeks; 10 animals/sex/dose) duration each :

Group 1(Control): Vehicle control

Group 2 (Low dose): 10 mg/kg/day

Group 3 (Mid dose): 100 mg/kg/day

Group 4 (High dose): 500 mg/kg/day

All animals were inspected daily for mortality and moribundity, for signs of ill health or overt toxicity and water consumption. Detailed clinical examination, body weight and food consumption were performed on a weekly basis. Ophthalmic examinations were done prior to initiation of treatment and during Weeks 4 and 13. Blood and urine samples were collected from all animals designated in Week 4, from all surviving animals in Week 13, and from the 5 male and 5 female animals from each group with the highest identification numbers surviving to the end of the treatment-free period for clinical laboratory examinations (hematology and clinical chemistry and urinalysis).

All designated animals were euthanized by an intraperitoneal injection of sodium pentobarbitone and exsanguinated after 5 (interim sacrifice), 14 (terminal sacrifice) and 19 weeks (sacrifice after treatment free period). Adrenals, kidneys, ovaries, testes, brain, liver and spleen were weighed. Slides of selected tissues preserved during necropsy were prepared (Hematoxylin and Eosin staining) for all tissues from control and high dose groups and all gross lesions from animals sacrificed at Week 4 were examined for microscopic alterations. Kidneys from all animals in control and high dose groups and all gross lesions from all animals sacrificed in Weeks 13 and 18 were similarly prepared and examined. In addition the kidneys from group 3 male animals were also examined.

One high dose male animal died of multifocal chronic myocarditis during Week 9 and one high dose male died during the treatment free period. Renal tubular vacuolation and basophilia with moderate multifocal cortical mineralization was considered to be the cause of death of the latter animal. There were no treatment-related changes on clinical observations, body weights and body weight changes, food consumption, water consumption, ophthalmic findings, heamotology and clinical chemistry at any dose level tested. However, urinalysis revealed increased levels of zinc and copper in high dose group at the Week 4 and 13 analyses. Levels of urinary iron were also increased in high dose females at the Week 4 analysis. Although the individual values were variable there were slight effects on zinc and copper levels still apparent after the treatment-free period (19 week). The increased amounts of zinc, iron and copper ions in the urine of the treated animals may be related to the histopathological lesions in kidney.

There were slight increases in male mean relative kidney weights in the high dose group at the Week 4 necropsies and Week 13 necropsies, when compared to respective controls. This increase in relative kidney weights of high dose group males may be related to histopathological lesions in kidney. By the end of the treatment free period, this effect was not apparent. Although there were no treatment related macroscopic findings in the Week 4 but at Week 13, high dose males (3 animals) had diminished seminal vesicles and 1 male had enlarged kidneys. The treatment related changes associated with test substance involve the kidney, suggesting kidney as the target organ.

Treatment-related microscopic changes characterized by renal tubular vacuolation with a slight increase in the incidence of basophilic tubules were noted in high dose male animals after 13 weeks of treatment. With the exception of one high dose male, which died during the treatment free period (19 week) and had marked tubular vacuolation of the kidneys, no microscopic observations related to treatment were noted after the 4 week interim sacrifice. Further, the minimal to moderate vacuolation appeared reversible as it was not apparent after 4 weeks without treatment.

In conclusion, oral administration of 2 -hydroxyethyliminodiacetic acid toCrl:CD(SD)BR rats at dose levels of 0, 10, 100 and 500 mg/kg bw/day in drinking water up to 13 weeks revealed a no observed effect level (NOEL) of 100 mg/kg bw/day (nominal) and 41 mg/kg bw/day (actual) based on reversible pathological lesions of kidney observed in high dose group animals).

This repeated dose toxicity study is classified as acceptable, and satisfies the guideline requirements of OECD 408 method.