Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 1995 - March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Batch No. is given, EU Method B.26 is followed (equivalent to OECD guideline 408) and study is performed according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

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)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

EC Number:

283-041-9

EC Name:

Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

Cas Number:

84539-53-7

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

non specified (UVCB substance)

Constituent 2

Reference substance name:

EDDHMA-Fe

IUPAC Name:

EDDHMA-Fe

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Bolikel FE, FeEDDHMANa, N,N'-Bis(2-hydroxy-4-methylphenyl)ethylenediamine diacetic acid, ferric-sodium complex
- Physical state: red brown powder
- Analytical purity: 65%
- Impurities (identity and concentrations): no info
- Purity test date: 16 and 20 November 1995
- Lot/batch No.: 954243 d.d. 19-10-1995
- Expiration date of the lot/batch: not indicated
- Stability under test conditions: stable under storage conditions; stable in water for at least 96 hrs
- Storage condition of test material: at room temperature, dry in the dark; during the test formulations at ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: at start of treatment approximately 6 weeks
- Weight at study initiation: mean 205 g (males), mean 169 g (females) at Day 1 of treatment
- Fasting period before study: no info, except that animals were fasted overnight prior to necropsy
- Housing: 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors
- Diet (e.g. ad libitum): free access to standard pelleted laboratory animal diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (fluctuations not have affected study integrity)
- Humidity (%): 50 (fluctuations not have affected study integrity)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day


IN-LIFE DATES: From: November 28, 1995 To: February 28, 1996 (males); March 1, 1996 (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily immediately prior to dosing. Based on results of previous studies in rats, the dose levels for the 90-day toxicity study were selected to be 0, 20, 100 and 500 mg/kg/day.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable (distilled water)
- Concentration in vehicle: 0, 4, 20 and 100 mg/ml (or mg/g)
- Amount of vehicle (if gavage): dose volume is 5 ml/kg bw
- Lot/batch no. (if required): not required
- Purity: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of pretreatment, week 2 and week 13 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations). In addition, stability over 4 hours was confirmed at the pretreatment analysis.
No acceptability criteria were given, so a range within 90-100% is adopted.
- Pretreatment: all samples were accurate enough (within 90-100%), except one which was 88%. The ‘relative (%) to mean concentration’ for homogeneity were all within 90 – 100%. And the relative difference (%) for stability was maximal 2%. Formulations were shown to be stable for at least 4 hours.
- Week 2 and week 13 formulations: concentrations as well as homogeneity represented an acceptable level for formulations.

Duration of treatment / exposure:

90 days (13 weeks)

Frequency of treatment:

Once daily for at least 90 days, approximately the same time each day, 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 100, and 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of previous studies in rats, the dose levels for the 90-day toxicity study were selected to be 0, 20, 100 and 500 mg/kg/day.
- Rationale for animal assignment (if not random): not applicable (random)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no info

Positive control:

Not needed

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations and time schedule: mortality / viability twice daily; clinical signs at least once daily (time of onset, degree and duration were recorded)

DETAILED CLINICAL OBSERVATIONS:No
- Time schedule: not applicable

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and on the day preceding the first necropsy date

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: 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 AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: no quantitative investigation

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined and time schedule for examinations: at pre-test all animals; at week 13 groups 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were collected under light ether anaesthesia, between 7:30 and 9:30 a.m.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight before blood sampling, but water was provided
- How many animals: all rats/sex/group
- Parameters checked: Erythrocyte count (RBC), Haemoglobin (HB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin concentration (MCHC), Platelet count, Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count (neutrophils, SEG; eosinophils, EO; basophils, BASO; lymphocytes, LYMPH; monocytes, MONO), Prothrombin time (PT), Partial thromboplastin time (PTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes
- How many animals: all rats/sex/group
- Parameters checked: Alanine aminotransferase (ALAT/GPT), Aspartate aminotransferase (ASAT/GOT), Bilirubin total (BILI T), Cholesterol total (CHOLEST T), Triglycerides (TRIGL), Creatinine, Glucose, Urea, Protein total (PROTEIN T), Protein albumin (ALBUMIN), Protein globulin (GLOBULIN), Albumin Globulin ratio (A/G RATIO), Alkaline phosphatase (ALP), Sodium, Potassium, Chloride, Calcium, Phosphorus (INORG PHOSPH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals surviving to the end of the observation period were fasted overnight prior to necropsy, deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions o f all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, Aorta, Brain, Caecum, Cervix, (Clitoral glands), Colon, Duodenum, Epididymides, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including knee joint, Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), (Nasopharynx), Oesophagus, Ovaries, Pancreas, Pituitary gland, (Preputial gland), Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.
HISTOPATHOLOGY: Yes, Slides of all tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions and lungs of all animals (all dose groups) were examined by a pathologist. Based on the treatment related morphologic changes, kidneys were also examined from all rats of the intermediate dose groups. Tissues mentioned between brackets were not examined.

Other examinations:

No.

Statistics:

The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The exact Fisher-test was applied to the ophthalmoscopic observations.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Clinical signs: Changes noted among animals receiving 500 mg/kg/day included lethargy, hunched posture, piloerection and/or emaciation. The incidences and duration of these clinical signs varied over the study period and the majority of symptoms was observed during the second part of the treatment period.
In addition, hunched posture was observed in one female of the 100 mg/kg dose group on a few occasions. No further signs of toxicity or behavioural changes were noted among treated animals.
Excessive salivation was seen among a few animals of the 500 mg/kg/day dose group. This is often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or the taste of the test substance.
Red discolouration of the excreta was ascribed to the staining properties of the test substance. Therefore these findings were considered not to be signs of systemic toxicity.
Other findings noted in control and treated animals were considered to be within the range of biological variation for rats of this age and strain or were related to blood sampling procedures.
Mortality: No mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of males and females receiving 500 mg/kg/day were decreased during the study period, attaining statistical significance in most cases.
No effects on body weights were seen among animals receiving 20 or 100 mg/kg/day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was slightly decreased in males treated at 500 mg/kg/day throughout the study period. Relative food consumption values remained within the range of normal variation. No clear changes in food consumption were noted in females receiving 500 mg/kg/day and in animals treated at 20 or 100 mg/kg/day.

FOOD EFFICIENCY
Not examined.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No data.

OPHTHALMOSCOPIC EXAMINATION
There were no significant changes noted in ophthalmology findings at week 13 when compared to pretest examination that were attributed to treatment with FeEDDHMANa.

HAEMATOLOGY
Red blood cell count, haemoglobin and haematocrit values were decreased in males and females receiving 500 mg/kg/day and in males receiving 100 mg/kg/day. In addition, mean cellular volume was slightly increased among 500 mg/kg treated males and red cell distribution width was decreased in 100 or 500 mg/kg treated males.
Increased total platelet counts were noted in males and females of the 500 mg/kg/day dose group.
Other statistically significant differences arising between controls and treated animals were considered to have arisen by chance and not to represent a change of biological significance as no dose relationship was seen or no concurrent changes were noted in the opposite sex.

CLINICAL CHEMISTRY
Treatment-related effects were observed for total cholesterol, creatinine and urea levels of animals receiving 500 mg/kg/day.
An increase in group mean creatinine was seen in males and a slight increase in cholesterol was noted among males and females. Serum urea was increased in females.
Both creatinine and urea elevations among females were mainly attributable to very high values in one single animal (animal 73).
Other values in treated animals achieving a level of statistical significance when compared to controls, were considered not to represent a clear sign of toxicity. Changes were of no biological relevance and/or resulted from slightly high control values (ALAT, ASAT). Furthermore, values remained within the range of historical data and no corroborative findings in the opposite sex were seen.

URINALYSIS
Not examined.

NEUROBEHAVIOUR
Not examined.

ORGAN WEIGHTS
Autopsy body weights were decreased in males and females receiving 500 mg/kg/day.
Kidney weights and kidney:body weight ratios were increased in males and females treated at 500 mg/kg and in males treated at 100 mg/kg.
Weights of testes, brain and heart are relatively independent of changes in body weight. Therefore, increased organ:body weight ratios, noted for these organs in animals receiving 500 mg/kg/day, were considered to be an indirect effect of lower body weights at autopsy and therefore not to represent a sign of toxicity.
Slightly higher liver:bodyweight ratios among 500 mg/kg treated females were considered to be within the range of normal variation for rats in a study of this duration.
Other statistically significant changes in organ weights or organ:body weight ratios were considered to have occurred by chance, as no dose relationship was seen and no concurrent changes were observed in the opposite sex.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Findings noted among treated and control animals were considered to be within the range of biological variation for rats of this age end strain or related to blood sampling procedures and therefore not to represent a change of toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys of some male and female rats treated at 500 mg/kg/day showed degenerative changes which were characterized by cortical tubular cell vacuolation.
The small number of other changes recorded in control and treated animals were within the range commonly seen for rats of this age and strain.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data.

HISTORICAL CONTROL DATA (if applicable)
No data.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

other: increased relative kidney weight, nephrotoxic effects (cortical tubular cell vacuolation), and changes in haematology and clinical chemsitry parameters in animals receiving 500 mg/kg bw/day

2

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that oral administration of FeEDDHMANa to rats for thirteen weeks resulted in changes in the kidneys of rats which had received 500 mg/kg bw/day; these consisted of histopathological changes, increased relative kidney weight, increases in blood urea, total cholesterol and creatinine concentrations and decreases in erythrocyte count, haemoglobin, haematocrit and red cell distribution width. These changes were corroborated by a decrease in body weight gain. Males receiving 100 mg/kg bw/day, showed a slight increase in relative kidney weight and slight decreases in erythrocyte count, haemoglobin, haematocrit and red cell distribution width. Therefore, the level of 100 mg/kg/day was considered a No-Observed-Effect Level (NOEL) for female rats and the level of 20 mg/kg/day was considered a No-Observed-Effect Level (NOEL) for male rats. In view of the absence of histopathological kidney effects in males treated with 100 mg/kg bw, and in view of the slight haematological changes, the NOAEL in male rats was considered to be close to 100 mg/kg bw.

Executive summary:

The study was designed to investigate the systemic toxicity of the test material and complies the recommendations of the OECD Guidelines for Testing of Chemicals No. 408. The test material was administered by oral gavage to three test groups, each consisting of 10 male and 10 female Wistar rats, for thirteen weeks, at dose levels of 20, 100 or 500 mg/kg bw/day. One control group of 10 male and 10 female rats was dosed with the vehicle (distilled water). Clinical signs were evaluated daily. Bodyweight and food consumption were monitored during the study. Ophthalmoscopy, haematology and clinical chemistry were also evaluated. All animals were subjected to a gross necropsy examination and a comprehensive histopathological evaluation of tissues from test and control groups was performed. In addition, organ weights were determined.

Test substance preparations in distilled water appeared to be stable and homogeneous and sufficiently accurate concentrations were encountered for the purpose of this study.

There were no deaths. The general health status of animals receiving FeEDDHMANa for 90 days at a dose level of 500 mg/kg/day was affected as reflected in clinical observations and retarded growth.

The kidney appeared to be an important target organ. Renal effects were mainly seen in 500 mg/kg treated animals and included cortical tubular cell vacuolation, changes in clinical biochemistry parameters as creatinine, urea and cholesterol, and increased kidney weights. Latter finding was also noted in 100 mg/kg treated males.

It was noted that the increased urea and creatinine values for females were attributable to high values in one single animal only.

Haematological changes comprised decreased values for total red blood cell count, haemoglobin, haematocrit and red cell distribution width in animals receiving 100 mg/kg/day (males) or 500 mg/kg/day (males and females); in males a dose-response relationship was obtained. Increased platelet counts were seen in 500 mg/kg treated animals.

Overall, oral administration of FeEDDHMANa to rats for thirteen weeks evoked changes in the kidneys of rats which had received 500 mg/kg bw/day; these consisted of histopathological changes, increased relative kidney weight, increases in blood urea, total cholesterol and creatinine concentrations and decreases in erythrocyte count, haemoglobin, haematocrit and red cell distribution width. These changes were corroborated by a decrease in body weight gain. Males receiving 100 mg/kg bw/day, showed also an increase in relative kidney weight and decreases in erythrocyte count, haemoglobin, haematocrit and red cell distribution width. Therefore, the level of 100 mg/kg/day was considered a No-Observed-Effect Level (NOEL) for female rats and the level of 20 mg/kg/day was considered a No-Observed-Effect Level (NOEL) for male rats.

In view of the absence of histopathological kidney effects in males treated with 100 mg/kg bw, and in view of the slight haematological changes, the NOAEL in male rats was considered to be close to 100 mg/kg bw.