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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

2000-08-29 to 2000-10-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC, directive No. 96/54, B7, 30th September 1996

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA, guideline 799, 9620-62-128, 15th August 1997

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): EDDHAS Fe, sel de K.
- Molecular formula (if other than submission substance): C18H14FeN2O12S2(3K)
- Molecular weight (if other than submission substance): 687.3
- Smiles notation (if other than submission substance): O=C(O)C(NCCNC(C(=O)O)c1cc(ccc1(O))S(=O)(=O)O)c2cc(ccc2(O))S(=O)(=O)O
- InChl (if other than submission substance): InChI=1S/C18H20N2O12S2/c21-13-3-1-9(33(27,28)29)7-11(13)15(17(23)24)19-5-6-20-16(18(25)26)12-8-10(34(30,31)32)2-4-14(12)22/h1-4,7-8,15-16,19-22H,5-6H2,(H,23,24)(H,25,26)(H,27,28,29)(H,30,31,32)
- Structural formula attached as image file (if other than submission substance): see Fig. below
- Substance type: Fe chelate
- Physical state: brownish/red powder
- Analytical purity: 55±2%
- Impurities (identity and concentrations): none
- Purity test date: 2000-10-24
- Expiration date of the lot/batch: 2001-01-24
- Stability under test conditions: at least 6 months from receipt (date of receipt: 24 July 2000)
- Storage condition of test material: room temperature and protected from humidity
- Other:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
44 Sprague-Dawley rats (22 males and 22 females) were received at test facility.
- Source: Iffa Credo, L'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: 196 g (range: 182 g to 209 g) for the males and 156 g (range: 137 g to 175 g) for the females
- Fasting period before study: no
- Housing: individually in suspended wire-mesh cages (43.0 cm x 21.5 cm x 18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage.
- Diet (e.g. ad libitum): free access to A04 C pelleted maintenance diet, batch No. 00331 (UAR, Villemoisson, Epinay-sur-Orge, France. Prior blood sampling the animals were fasted overnight.
- Water (e.g. ad libitum): filtered tape water
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2,
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water, obtained by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in the required quantity of vehicle in order to achieve the concentrations of 30, 90 and 200 mg/mL and then homogenized using a magnetic stirrer.
The test substance dosage forms were made up to 4 days from days 1 to 3 and up to 9 days, from day 4 of treatment, and were stored at +4°C prior to use. The dosage forms were delivered each day to the animal room.

VEHICLE

- Concentration in vehicle: 30, 90 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dosage forms
Before the start of treatment, the suitability of the proposed dosage form preparation procedure was determined by the analysis of stability of dosage forms which were prepared using this procedure. During the treatment period, the concentration of the test material was checked in dosage forms prepared for use in the study.

Stability
Two dosage forms were prepared to evaluate the stability:
- a dosage form at low concentration (2 mg/mL),
- a dosage form at high concentration (200 mg/mL). Each dosage form was analysed immediately after preparation and was then stored at 4°C (protected from light) and sampled after 4 and 9 days storage. Samples taken on days 4 and 9 were analyzed as soon as possible after sampling.


Concentration
The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1 and 4 was determined.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 450 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected on the basis of the results of a 7-day range-finding toxicity study by oral route performed in the same species (CIT/Study No. 20508 TSR) in which no clinical signs or macroscopic findings were noted in any treated groups. Consequently the same dose-levels were selected.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day for mortality or signs of morbidity and at least once a day for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then at the end of weeks 1, 2, 3 and 4 (at least 12 hours after the last treatment. All animals of each group were observed in the cage, in the hand and in the standard arena, by observers unaware of the animal's treatment. Detailed clinical observation in week 4 was performed before blood sampling.
The following parameters were assessed:
- in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypic behaviour and breathing, ataxia, hypotonia.

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation of the animals into groups, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION (gavage study):
The quantity of food consumed by the animals of each cage was recorded once a week, over a 7-day period, until the end of the study (calculated as mean values in g/animal/day)

OPHTHALMOSCOPIC EXAMINATION: No (pupil reflex was examined: see Neurobehavioural Examination)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: all animals
- Parameters were examined:
Erythrocytes (RBC)
Hemoglobin (HB)
Mean Cell Volume (MCV)
Packed Cell Volume (PCV)
Mean Cell Hemoglobin Concentration (MCHC)
Mean Cell Hemoglobin (MCH)
Thrombocytes (PLAT)
Leucocytes (WBC)
Differential White Cell count with cell morphology: neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M)
Prothrombin Time
Activated Partial Thromboplastin Time
Fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of week 4
- Animals fasted: Yes, prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours
- How many animals: all animals
- Parameters were examined:
Sodium (Na+), Potassium (K+), Chloride (C1-), Calcium (Ca++)
Inorganic phosphorus (I.PHOS) Glucose (GLUC)
Urea (UREA)
Creatinine (CREAT)
Total Bilirubin (TOT.BIL)
Total Proteins (PROT)
Albumin (ALB)
Albumin/globulin ratio (A/G)
Cholesterol (CHOL)
Triglycerides (TRIG)
Alkaline phosphatase (ALP)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals were evaluated before the first day of treatment, and then at the end of week 4, at least 12 hours after the last treatment. The observer performing the evaluation was not aware of the treatment group of the animal.
Reactivity to manipulation or to different stimuli in week 4 was performed before blood sampling.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity (was measured by automated infra-red sensor equipment recording individual animal activity over 30-min period, before the first day of treatment and then in week 4)/ other: pupil reflex, visual stimulus, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the tissue procedures table (please refer to table in "Any other information on materials and methods incl. tables" for animals of the control and high-dose groups (groups 1 and 4) killed at the end of the treatment period,
all macroscopic lesions of all the animals of the low- and intermediate- dose groups (groups 2 and 3) killed on completion of the treatment period.

Other examinations:

ORGAN WEIGHTS
The organs specified in the Tissue Procedures Table ((please refer to table in "Any other information on materials and methods incl. tables") were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Statistics:

Scheme of statistical analysis is attached (see below)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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
No mortalities and no treatment-related clinical signs were noted.

BODY WEIGHT AND WEIGHT GAIN
The mean body weight gain of males and females given the test substance at 150, 450 and 1000 mg/kg/day was similar to that of respective control groups throughout the treatment period (males: +158 g, +139 g and +145 g vs. +146 g in controls; females: +66 g, +74 g and + 76 g vs. +70 g in controls

FOOD CONSUMPTION
The mean food consumption of males and females given the test substance was similar to that of respective control groups throughout the treatment period.

HAEMATOLOGY
No toxicologically significant changes were noted

CLINICAL CHEMISTRY
No treatment-related differences from controls were noted in the treated animals for all the parameters examined.

NEUROBEHAVIOUR
No specific signs of a neurotoxic action of the test substance were noted.

ORGAN WEIGHTS
No treatment-related effects were noted in the organ weights.

GROSS PATHOLOGY
The following necropsy findings were noted in the digestive tract:
blackish contents were noted in several parts of the digestive tract of treated animals as follows:
- stomach, in 1/5 males given 1000 mg/kg/day,
- ileum in 1/5 females given 450 mg/kg/day and in 1/5 males given 1000 mg/kg/day,
- cecum, in 1/5 females given 150 mg/kg/day, in 4/5 males and all the females given 450 mg/kg/day and in 4/5 males and all the females given 1000 mg/kg/day,
- colon, in 1/5 females given 150 mg/kg/day, in 3/5 males and 4/5 females given 450 mg/kg/day and in 3/5 males and 1/5 females given 1000 mg/kg/day,
- rectum, in 1/5 males given 450 mg/kg/day and in 1/5 males given 1000 mg/kg/day.
Microscopic examination revealed no changes that related to this blackish material which was consequently considered to be a remnant of the test substance and therefore of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
No changes were seen that were related to treatment.

OTHER FINDINGS
No other findings

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were noted in any parameter tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Chemical Analyses of the Dosage Forms

Stability

The results of the analyses demonstrated a satisfactory stability of the two dosage forms over a 9-day period at +4°C (protected from light).

Concentration

Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the dosage forms administered since the deviations from nominal concentration were in the requested range of +10%.

Applicant's summary and conclusion

Conclusions:

The daily administration of the test substance, EDDHAS Fe 3K (batch No. CED 0701), at the dose-levels of 150, 450 and 1000 mg/kg/day, by oral route (gavage) to rats for 28 days did not produce any signs of toxicity.
Consequently, under our experimental conditions, the No Observable Effect Level (NOEL) was established at 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test substance, EDDHAS Fe 3K, following daily oral administration (gavage) to Spague-Dawley rats for 28 days.

Methods

Groups of five males and five females Sprague-Dawley rats received the test substance, EDDHAS Fe 3K, daily by gavage at the dose-levels of 150, 450 or 1000 mg/kg/day for 28 days. An additional group of five males and five females received the vehicle alone (purified water) under the same experimental conditions, and acted as a control group. The animals were checked daily for mortality and clinical signs. A functional observation battery was performed on all animals of each group, once in predose and then in week 4; in addition, a detailed clinical observation was performed on all animals of each group, once a week until the end of the treatment period. Motor activity was recorded on all animals once in predose and in week 4. Body weight and food consumption were recorded once a week. Hematological and blood biochemical investigations were performed for all animals at the end of the treatment period. On completion of the treatment period, all animals were killed and submitted to a complete macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on designated tissues of the animals of the control and high dose-level group.

Results

Mortality

No unscheduled deaths occurred during the study.

Clinical signs

No clinical signs of toxicological significance were observed in any treated animals.

Functional observation battery and motor activity

No specific signs of a neurotoxic action of the test substance were noted.

Body weight and food consumption

Overall body weight gains were similar in control and treated groups and food consumption was considered to be unaffected by treatment.

Hematology and blood biochemistry

No changes of toxicological significance were noted in any parameters.

Organ weights

No notable differences in organ weights were noted between control and treated groups.

Macroscopic and microscopic examinations

No changes of toxicological importance were observed.

Conclusion

The daily administration of the test substance, EDDHAS Fe 3K, at the dose-levels of 150, 450 and 1000 mg/kg/day, by oral route (gavage) to rats for 28 days did not produce any signs of toxicity. Consequently, under our experimental conditions, the No Observable Effect Level (NOEL) was established at 1000 mg/kg/day.