Ammonium iron(III) trimethylenediaminetetraacetate hemihydrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 1988 - 4 January 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was peformed according to OECD guidelines and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

2 week recovery group was added.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium iron(III) trimethylenediaminetetraacetate hemihydrate

EC Number:

400-660-3

EC Name:

Ammonium iron(III) trimethylenediaminetetraacetate hemihydrate

Cas Number:

111687-36-6

Molecular formula:

Hill formula: C11 H18 Fe N3 O8 CAS formula: C11 H14 Fe N2 O8.H4N

IUPAC Name:

iron(3+) ammonium 2-({3-[bis(carboxylatomethyl)amino]propyl}(carboxylatomethyl)amino)acetate hydrate

Details on test material:

Name: M-3175; 1,3-propylenediamine-N,N,N',N'-tetra acetic acid, ammonium iron (+3) salt
Appearance: yellow powder
Batch No.: VG-01
Purity: 99%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (U.K.), Margate, Kent, England
- Age at study initiation: four to five weeks old
- Weight at study initiation: 62 - 104 g
- Housing:The rats were housed in polypropylene cages (Pattern RCl, North Kent Plastics Ltd., Dartford, Kent, England; measuring 56 x 38 x 18 cm), with stainless steel mesh floors and lids. The cages were suspended in a battery capable of holding up to 21 cages, above absorbent crepe paper. The paper was changed two times per week; cages, cage-trays and water bottles were changed when necessary. Five rats of one sex were held in each cage.
- Diet (e.g. ad libitum): A commercially available complete pelleted laboratory rodent diet (LAD i, Labsure, Manea, Cambridgeshire, England) was available for the rats to consume ad libitum.
- Water (e.g. ad libitum): Drinking water was provided for the rats to consume ad libitum via two polyethylene bottles with chromium plated sipper-tubes.
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 21 November 1988 - 4 January 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations of the test material were prepared for administration as a series of graded concentrations in distilled water to provide the required dosages at a constant volume-dosage of 10 ml/kg bodyweight. Control rats received the vehicle (distilled water) alone at the same volume-dosage. All formulations were prepared freshly each day).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed formulations was determined by a trial preparation, made up as for Day 1 of treatment. Homogeneity.was determined by infra-red spectrophotometric assay of the trial formulations for the high and low dose groups immediately following their preparation, and stablilty was assessed by the same method after 24 and 48 hours storage at room temperature. The results indicated that the homogeneity and stability of M-3175 in distilled water were satisfactory.

Duration of treatment / exposure:

29 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
40, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 for the control- and high-dose group and 5 for the low- and mid-dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of toxicity data provided by a preliminary investigation. Three groups of three male and three female rats were given M-3175 once daily by-oral gavage for seven days at dosages of 200, 500 or 1000 mg/kg/day. At these dose levels there were no signs of treatment.
- Rationale for animal assignment (if not random): On arrival, the animals were assigned to cages according to a sequence of computer generated random numbers
- Rationale for selecting satellite groups: Studying reversibility of any effect
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): no data

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
A preliminary check for deaths or morbidity. At least two daily examinations for evidence of systemic toxicity or ill-health, the first
immediately before dosing and the second shortly after dosing.
An additional final check for systemic toxicity, or ill-health, on all full work days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed weekly examination including palpation.

BODY WEIGHT: Yes
- Time schedule for examinations: Each rat was weighed on the day that treatment commenced and twice weekly throughout the study period.

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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
The weight of food eaten by each cage of rats was calculated weekly by measurement of the amount of food given and that remaining in the food hoppers, together with an estimate of any food scattered.

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Water consumption was assessed visually in the course of daily observation. Quantitative
measurements were not undertaken.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (identity: no data)
- Animals fasted: Yes
- How many animals: all
- Parameters checked:
Packed cell volume (PCV), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Total Leucocyte count (WBC), Platelet count, Reticulocyte count.
In addition the following characteristics were recorded or calculated:
Leucocyte count (WBC), differential - Romanowsky stain and direct visual count, differentiating among neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B) and monocytes (M); also recording any normoblasts seen Mean cell haemoglobin concentration (MCHC) calculated
as Hb x 100 / PCV Mean cell volume (MCV)- calculated as PCV x 10 / RBC Mean cell haemoglobin (MCH) - calculated as Hb x 10 / RBC.
Using citrate as anticoagulant further samples were examined for: Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alkaline phosphatase (AP), Alanine amino-transferase activity (ALT), Aspartate amino-transferase activity (AST), Gamma glutamyl transpeptidase activity (GGT), Glucose concentration, Total bilirubin concentration, Total triglyceride concentration, Total cholesterol concentration, Total protein concentration, Electrophoretic protein fractions, Sodium (Na}, Potassium (K), Chloride (Cl), Calcium
(Ca) and Inorganic phosphorus (P)

URINALYSIS: Yes
- Time schedule for collection of urine: After three weeks of treatment (Day 25) each rat was placed in an individual metabolism cage for overnight urine collection under conditions of food and water deprivation.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearance, pH, Specific gravity (SG), Protein, Total reducing substances (Total red. subs.), Glucose, Ketones, Bilirubin, Urobilin, Nitrite, Blood, Microscopy the sediment from centrifugation was examined for epithelial cells (E), polymorphonuclear leucocytes (P), erythrocytes (R), crystals (C), spermatozoa and precursors (S) or other abnormalities (A).

NEUROBEHAVIOURAL EXAMINATION: No

Other:
Urine samples weer colelcted for each reversibility animal after ten days cessation of treatment (Day 39) and examined for:
Appearance (males only), Volume, pH, Specific gravity, Protein (males only), Blood (females only).
Blood samples were collected from each reversibility animal into EDTA or heparin anticoagulant after two weeks cessation of treatment (Day 44) and examined for:
Packed cell volume (PCV), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Alkaline phosphatase activity (AP), Aspartate amino-transferase activity (AST), Urea concentration, Total triglyceride concentration, Total cholesterol concentration (males only), Potassium (K) concentration (males only), Inorganic phosphorus concentration (P) (males only).
The measurement of chloride concentration in male and female reversibility rats was overlooked

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals were subjected to a detailed necropsy.

HISTOPATHOLOGY: Yes, Adrenals, Heart - auricle, - ventricle, Kidneys, Liver - two lobes, Spleen
The following tissues were not processed histopathologically but are held for possible future requirements: Brain, Eyes and optic nerves, Femoral bone and marrow, lungs (with main stem bronchi), Ovaries, Pituitary, Stomach, Testes, Thyroids (with parathyroids), Urinary bladder

Organ weights
After removal of adjacent fat and other contiguous tissue, the weights of the fonowing organs were recorded for each animal: Adrenals, Brain, Kidneys, Liver, Ovaries, Testes.

Statistics:

Inter-group differences in group mean bodyweight change, haematology, blood chemistry and urinalysis were evaluated by Student's 't'-test using a pooled variance. The results of this test are not presented for eosinophil, basophil or monocyte counts where the data are clearly not normally distributed. Urinary protein concentrations were assessed by the Mann Whitney test. Absolute and relative organ weights were analysed using Dunnett's test. Inter-group differences in the incidence of macro- Dr micropathological lesions were assessed by the Fisher Exact Probability test (two-tailed) .
Levels of statistical significance were chosen as p < 0.05 (a), p < 0.01 (b) and p < 0.001 (c) for 'Student's 't'-test and the Mann Whitney test, and p < 0.05 (a) and p < 0.01 (b) for Dunnetts test and the Fi scher Exact Probabil ity test. Inter-group differences that were not statistically significant (p > 0.05) are not annotated.

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:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no death. Signs of reaction to treatment were restricted to animals receiving 1000 mg/kg/day. They began as irregularly shaped,
moist faeces during the second week of treatment (Day 12). By the fourth week of treatment (Days 24~25) there was evidence of heavy perianal staining, loose faeces and dark stained faeces. The cage tray papers were observed to be dark stained on Days 17 to 19. Loose and dark stained faeces had resolved within two days and the perianal staining had resolved within four days of cessation of treatment. The appearance and behaviour of rats receiving 200 or 40 mg/kg/day were indistinguishable from the control animals.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was considered to have been unaffected by treatment with M-3175.
The bodyweight gain of male rats receiving 1000 mg/kg/day was slightly inferior to the male control animals during the treatment period and slightly superior (p < 0.05) during the reversibility period. However, the differences were small and, in the absence of simiiar observations in females receiving this dosage, they could not be unequivocally ascribed to an effect of the test material.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was considered to have been unaffected by treatment with M-3175. The reduced food consumption values recorded for all groups of rats during Week 4 of treatment and Week 2 of the reversibilty period, compared with preceding values, resulted from the overnight withdrawal of food for collection of blood and urine

FOOD EFFICIENCY
Food utilisation efficiency was considered to have been unaffected by treatment with M-3175.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Visual inspection of water bottle residues did not reveal any evidence of inter-group differences in water consumption.

HAEMATOLOGY
After four weeks of treatment, the packed cell volume (p <-0.01), haemoglobin concentration {~< 0.05) and red cell numbers (p > 0.05) of male rats receiving 1000 mg/kg/day were slightly higher than those of the male control animals. Similar, very small differences in these parameters were also recorded at this time between female rats receiving this dosage and the female controls. After two weeks cessation of treatment, the erythrocyte characteristics of the reversibility animals that received 1000 mg/kg/day were essentially the same as those of the control animals. The differences in the lymphocyte and total leucocyte numbers between male rats treated at 1000 mg/kg/day and the male control animals (p < 0.05 ) were considered to largely reflect a high control value, rather than an effect of M-3175. The small differences in platelet numbers and prothrombin time between female rats treated at 1000 mg/kg/day and the female controls (p < 0.05) were not considered to be biologically significant. The haematology of rats treated at 200 or 40 mg/kg/day was considered to have been undisturbed.

CLINICAL CHEMISTRY
Animals of both sexes receiving 1000 mg/kg/day displayed lower alkaline phosphatase (p < 0.001) and aspartate amino-transferase activities (p < 0.01 for males and p < 0.05 for females), slightly lower urea (p > 0.05 for males and p < 0.01 for females) and higher triglyceride concentrations (p < 0.001 for males and p > 0.01 for females), and slightly lower chloride levels (p < 0.001), than those of their respective control animals. In addition, males treated at this dosage showed slightly lower potassium (p < 0.05) and inorganic phosphorus levels (p < 0.01), and slightly higher cholesterol (p < 0.05) concentrations, than the male control animals. Plasma concentrations of chloride ions (p < 0.001) and inorganic phosphorus (p < 0.05) in male rats treated at .200 mg/kg/day and phosphorus(p < 0.05) in males receiving 40 mg/kg/day were similarly lower than those of the controls. There was no other evidence to suggest an effect of treatment in rats receiving 200 or 40 mg/kg/day. Since the reductions in plasma phosphorus concentrations were not clearly dosage-related, and there was no indication of any similar effect in females even .at the highest dosage, it is considered that the inter-group differences were fortuitous and unrelated to treatment with M-3175. This view is supported by persistence of low plasma phosphorus concentrations (p < 0.05) after respite from treatment in males formerly receiving 1000 mg/kg/day, when compared with controls. Recovery in respect of effects on alkaline phosphatase and aspartate amino-transferase (incomplete in females) activities, urea, triglyceride, cholesterol and potassium concentrations was clearly apparent

URINALYSIS
After three weeks of treatment, the urine of animals of both sexes receiving 1000 mg/kg/daywas slightly lower in volume (p > 0.05 for males and p < 0.01 for females) and pH (p < 0.05 for males p < 0.001 for females) and was of slightly higher specific gravity (p<0.05 for females and p<0.001 for females than the value recorded for the control animals. Many samples collected at this time from the male rats receiving 1000 mg/kg/day were contaminated by loose faeces and could not be analysed. Most of the other samples from these animals, which were not overtly contaminated, were dark yellow in colour and had generally sl ightly higher protein concentrations than in control samples. These changes, and the presence of blood pigment in the urine of one female receiving 1000 mg/kg/day, are considered to reflect a degree of faecal contamination that was insufficient to preclude analysis. After ten days respite from treatment, the urine of rats previously receiving 1000 mg/kg/day was similar to that of the control animals. The urine of rats treated at 200 or 40 mg/kg/day was considered to have been unaffected by M-3175.

ORGAN WEIGHTS
Organ weights were considered to have been unaffected by treatment with M-3175.

GROSS PATHOLOGY
There was no macroscopic change which was attributed to treatment with M-3175.

HISTOPATHOLOGY
There were no microscopic changes of pathological significance in animals killed after four weeks of treatment, which were attributed to administration of M-3175.


HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 1000 mg/kg bw/d: mild, reversible effects, principally associated with disturbance of intestinal function. 200 mg/kg bw/d: slightly lower plasma chloride levels in the males only. 40 mg/kg bw/d: no effects. NOAEL: 200 mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that daily oral administration of M-3175 to rats at a dosage of 1000 mg/kg/day elicited mild, reversible effects, principally associated with disturbance of intestinal function. The NOAEL of administration was considered to be 200 mg/kg/day.

Executive summary:

Three groups of five male and five female CD rats were treated with M-3175 by oral gavage at dosages of 40, 200 or 1000 mg/kg/day for four consecutive weeks. The test material was administered in distilled water, at a volume-dosage of 10 ml/kg. A similarly constituted group of rats received the vehicle alone at the same volume-dosage and acted as a contemporaneous control. An additional five male and five female rats were allocated to each of the control and high dosage groups and were retained for a further two weeks without treatment, to assess the reversibility

of any induced effects. At the end of the treatment or reversibility periods the animals were humanely killed and subjected to a aetailed necropsy.

Selected tissues were taken and processed for microscopic examination.

There was no death. Effects of treatment in rats receiving 1000 mg/kg/day were: loose, black stained faeces and heavy perianal staining by Week 4

slightly higher packed cell volume, haemoglobin concentration and erythrocyte numbers, compared with the control animals lower plasma alkaline phosphatase and aspartate amino-transferase activities, slightly lower urea concentrations, higher triglyceride concentrations and slightly lower chloride levels than the control animals. Males also showed slightly lower potassium levels and slightly higher cholesterol concentrations, than the male controls slightly lower urinary volume and pH and slightly higher specific gravity, than the control animals. Urine samples from the male rats were contaminated by faeces or were dark yellow. Most of these effects resolved during the two-week reversibility period.

Differences between rats receiving 200 mg/kg/day and the control animals that may have reflected an effect of treatment were confined to slightly lower plasma chloride levels in the males only.

There-were no effects of treatment rats treated at 40 mg/kg/day. There were no micropathological changes which were attributed to the administration of M-3175.

It is concluded that daily oral administration of M-3175 to rats at a dosage of 1000 mg/kg/day elicited mild, reversible effects, principally associated with disturbance of intestinal function. The NOAEL of administration was considered to be 200 mg/kg/day.