Sodium [N-[2-[bis(carboxymethyl)amino]ethyl]-N-(2-hydroxyethyl)glycinato(4-)]ferrate(1-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

April-August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

yes

Remarks:

for histopathology (except for sex organs) 5 animals/sex/group were used

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

EC Number:

235-627-0

EC Name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

Cas Number:

12389-75-2

IUPAC Name:

iron(3+) sodium [{2-[{2-[bis(carboxylatomethyl)amino]ethyl}(carboxylatomethyl)amino]ethyl}(carboxymethyl)amino]acetate

Details on test material:

Chemical name: Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)
Purity: 11.6% (Fe content)
Batch no: CFC 9883
Expiry date: 1 January 2013

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutshland, Sulzfeld, Germany
- Age at study initiation: 5 weeks (females), 6 weeks (males)
- Weight at study initiation: mean weight males ca. 170 g; mean weight females ca. 106 g
- Fasting period before study: not applicable
- Housing: 4 per sex in macrolon cages, with wood shavings as bedding material, and paper strips as environmental enrichment
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 degrees C, reaching a minimum of 19.2 degrees C
- Humidity (%): at least 45% and not exceeding 65%. During several periods, humidity was outside the limits reaching a minimum of 43% and a maximum of 96% during a short period
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 April to 4 August 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulations was performed one day before the first day of the dosing period and at weekly intervals thereafter until completion of the dosing phase of the study. The different concentrations of the test substance in tap water were prepared by stirring on a magnetic stirrer for at least 1h. The pH of the test formulations of groups 2, 3 and 4 were set between pH 6-7 using sodium carbonate (Na2CO3). Subsequently, under continuous stirring, 8 aliquots (7 days plus 1 extra) were taken according to the volume required for each dosing. Aliqouts were stored in a refrigerator in the dark. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire administration period in order to maintain the homogeneity of the test substance in the vehicle.
Sodium carbonate was added to all three test formulations to adjust the acidity of the formulations to pH 6-7: it appeared that the amount of test substance used for the preparation of the test solution for the high-dose group did not dissolve unless the pH was adjusted. On the first 2 days of the study, animals of the low- and mid-dose groups were treated with test formulations without the addition of sodium carbonate. From Day 2 onwards, all animals of the low-, mid- and high-dose groups were treated with test formulations with added sodium carbonate. In week 2 of the study, the pH of the test formulations of the low-, mid- and high-dose groups were marginally higher (resp 7.03, 7.05 and 7.06). This also applied for week 8 of the study, regarding test formulations of the low- and mid-dose groups (resp 7.18 and 7.01).

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To determine the homogeneity and content of DTPA-FeNaH in gavage liquid, iron was used as a marker for the test item. Iron concentrations in gavage liquid were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES).
The concentrations of iron measured in the gavage liquids prepared on 15 April 2010, 15 June 2010 and 06 July 2010 were ‘close to intended’ (relative difference < 10 %) for all gavage liquids at all dose levels, except for the low-dose level gavage liquids prepared on 15 April 2010 (+10.7%) and the low-, mid- and high-dose level gavage liquids prepared on 06 July 2010 (+10.1%, +11.0% and +13.6%, respectively).

Duration of treatment / exposure:

10 weeks pre-mating, 16 days mating, 3 weeks gestation, and 4 days lactation

Frequency of treatment:

single daily application by gavage

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 500 and 1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on studies done with EDTA and EDTA-MnNa2
- Rationale for animal assignment (if not random): computer randomization proportionately to BW

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly during 10 weeks

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in week 8 of the pre-mating period
- Dose groups that were examined: all
- Battery of functions tested: FOB (including sensory activity and grip strength) and spontaneous motor activity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (males and females) and on day 1 and 4 of lactation (females)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: weekly (at same time as measurement of bw)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of week 10, prior to mating
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (water freely available)
- How many animals: 5 sex/group
- Parameters checked: haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of week 10, prior to mating
- Animals fasted: Yes (water freely available)
- How many animals: 5 sex/group
- Parameters checked: alkaline phosphatase activity (ALP), bilirubin (total), aspartate aminotransferase activity (ASAT), cholesterol (total), alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin (calculated), potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate (PO4), glucose (fasting)

URINALYSIS: No

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals as soon as possible after mating (at least 13 weeks of treatment)
- Maternal animals: All surviving animals at or shortly after day 4 of lactation (almost 14 weeks of treatment)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

ORGAN WEIGHTS:
- testes, epididymides (12 rats/group)
- adrenals, brain, heart, kidneys, liver, spleen, thymus (5 rats/sex/group)

HISTOPATHOLOGY:
- ovaries, uterus (12 rats/group; control and high dose group
- testes, epididymides, seminal vesicles, prostate, coagulating glands (12 rats/group; control and high dose group
- adrenals, axillary lymph nodes, brain, caecum, colon, femur, Peyer's patches, heart, kidneys, liver, lungs, mesenteric lymph nodes, peripheral nerve, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, urinary bladder (5 rats/sex/group; control and high dose group

Other examinations:

See at reproduction and developmental toxicity

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Haematology and clinical chemistry parameters were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.

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 examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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: Soft faeces were observed in males and females of the high-dose groups in various weeks of the pre-mating period . This finding was most pronounced in the first week of the study. Soft feaces were also observed in some rats of the mid-dose group in the first week of the study only. The findings in the high-dose group occasionally reached the level of statistical significance. The repeated occurrence of soft faeces in the high-dose group was considered treatment-related.
One female of the low-dose group (no. 33) was found dead on day 64 of the pre-mating period. In the fourth week of the study, one female of the mid-dose group (no. 57) showed a hunched body position, piloerection and was thin and weak. On Day 32 of the pre-mating period it was found dead. On Day 3 of the gestation period, a female of the high dose group (no. 85) was killed because it was thin and showed piloerection and abnormal respiration (rales, dyspnoea, decreased frequency). The death of females nos 33 and 85 is likely to have been caused by misdosing. The cause of death of
female no. 57 of the mid-dose group could not be examined at necropsy because its organs were already autolytic.

BODY WEIGHT AND FOOD CONSUMPTION: The mean body weight of males of the high-dose group was decreased when compared to the control group, reaching the level of statistical significance as from Day 35 of the study. In addition, the mean body weight change of males of the high-dose group was statistically significantly decreased at various time periods (Days 0-7, 28-49, 63-70). These observed effects on body weight were considered to be related to treatment. Food consumption was not affected by treatment.

TEST SUBSTANCE INTAKE: no effects (gavage)

WATER CONSUMPTION: not measured

OPHTHALMOSCOPIC EXAMINATION: not measured

HAEMATOLOGY: Prothrombin time was increased in males of the high-dose group. Haemoglobin concentration, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly increased in males of the high-dose group when compared to controls.

CLINICAL CHEMISTRY: Alkaline phosphatase activity (ALP) showed a dose-related and statistically significant decrease in males of the mid- and high-dose groups and in females of all treatment groups. In males of the high-dose group, alanine amino transferase activity (ALAT) was statistically
significantly decreased when compared to controls. The chloride concentration in males of the high-dose group was statistically significantly
decreased.

URINALYSIS: not measured

NEUROBEHAVIOUR: no treatment-related effects

ORGAN WEIGHTS: The relative weights of the kidneys and liver of male and female animals of the high-dose group were statistically significantly increased. These findings were considered treatmentrelated. The absolute weight of the epididymides was decreased in males of the mid- and high-dose
group. The relative weight of this organ was decreased in males of the high-dose group but not in males of the mid-dose group. Therefore, only the decreased weight of the epididymides of males of the high-dose group was considered to be related to treatment.

GROSS PATHOLOGY: no effects

HISTOPATHOLOGY (NON-NEOPLASTIC): no treatment-related effects

HISTOPATHOLOGY (NEOPLASTIC): no changes

HISTORICAL CONTROL DATA: not needed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

< 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose-related decrease in ALP levels (see at remarks)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Most of the effects observed were confined to animals of the high-dose group and comprised soft faeces (both sexes), decreased body weight gain (males), prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes). The soft faeces, and decreased body weight gain and decreased male fertility (see also at section 7.8.1) may point at zinc deficiency at the high concentration level. Since DTPA is a chelating agent like EDTA, DTPA depletes metals including zinc.

Alkaline phosphatase (ALP) activity in blood showed a dose-related decrease in males of the mid- and high-dose group and in females of all treatment groups. The toxicological relevance of this finding is not clear as increased serum ALP activity is generally considered to be a toxic effect. It has been shown that a decrease in ALP levels might also be the consequence of zinc deficiency or depletion of zinc.

The log K binding values for DTPA are as follows (for comparison the values of EDTA are also given):

DTPA: 16.2 (Fe(II)), 18.2 (Zn), 28.0 (Fe(III))

EDTA: 14.3 (Fe(II)), 16.5 (Zn), 25.1 (Fe(III))

[ A.E. Martell, R.M. Smith, NIST Critically selected stability constants of metal complexes; NIST standard reference database 46, Version 7.0, 2003)]

This means that DTPA (like EDTA) has the highest affinity for Fe(III), followed by Zn and then Fe(II). The gut enterocytes have the ability to convert Fe(III) to Fe(II) prior to absorption, so the Fe(III) is released, then converted to Fe(II) for which DTPA has a lower affinity so it picks up whatever else for which it has a higher affinity, such as zinc. Thus, the release of the Fe leaves the DTPA open to bind zinc either in the gut or subsequent to absorption (the absorption of DTPA is estimated to be ca. 5%, as with EDTA). This zinc chelation then causes the effects on male reproductive organs (see below) and also on the liver enzymes. In addition, ALP is an enzyme with 2 zinc atoms attached, one bound tightly and one not so much. Therefore, any chelator of zinc present in blood could remove some of the zinc or prevent sufficient zinc from being available to the enzyme. It is therefore concluded that the effect on serum ALP activity is most probably due to effects on zinc homeostasis. In addition, the removal of zinc has caused a decrease in ALP activity, it does not necessarily mean that the ALP level as such had decreased.

The toxicological significance of a decrease in serum ALP activity is not clear. On the one hand it can be expected that a sufficient deficiency of this enzyme in cells could lead to issues with DNA repair, cell replication, and other processes requiring the dephosphorylation of proteins, nucleotides, etc. On the other hand, it is not clear in how far a decrease in serum ALP activity reflects decreased cellular ALP activity. There are a number of diseases associated with decreased serum ALP levels such as severe anemia, hypothyroidism, malnutrition, hypophosphatasia, and chronic myelogenous leukemia. Most probably these diseases have resulted in decreased ALP activity rather than that decreased ALP activity has caused these diseases. In addition, it can be concluded that the body can cope with a decrease in serum ALP activity to a certain level, since in the present study there were no other effects observed at the mid- and low–dose level. As such it can be argued that the decrease in serum ALP activity is a treatment-related effect, but non-adverse.

Applicant's summary and conclusion

Conclusions:

It is concluded that at the low and mid dose the decrease in serum ALP activity is a treatment-related effect that is of questionable toxicological significance, and most probably not adverse in itself. Based on the above considerations, the No Observed Effect Level (NOEL) for parental toxicity is <150 mg/kg body weight/day whereas the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 500 mg/kg body weight/day.

Executive summary:

The objective of this study was to provide data on the possible effects of the test substance DTPA-FeNaH following subchronic exposure, and on reproductive performance of Wistar rats (see also section 7.8.1) and the development of pups (see also section 7.8.2) following daily oral administration at concentrations of 0, 150, 500 or 1500 mg/kg bw of the test substance by gavage to male and female rats during a pre-mating period of 10 weeks, during mating (16 days), and during gestation and lactation until postnatal Day 4 (PN Day 4).

The homogeneity and content of the test substance in the gavage solutions were confirmed by analysis.

Males and females of the high-dose group showed soft faeces in various weeks of the premating period. Daily clinical observations during the gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Mean body weights were decreased in males of the high-dose group from week 5 onwards. There were no treatment-related effects on female body weights during the entire study. No treatment-related effects were observed on food consumption of male and female animals during the entire study.

In males of the high-dose group, prothrombin time was increased, and haemoglobin concentration, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were statistically significantly increased. Alkaline phosphatase activity (ALP) showed a dose-related decrease in males of the mid- and high-dose group and in females of all treatment groups. In males of the high-dose group, alanine amino transferase activity (ALAT) and chloride concentration were decreased.

The relative weights of the kidneys and liver were increased in both sexes of the high-dose group. The relative weight of the epididymides was decreased in males of the high-dose group. Macroscopic examination at necropsy, and microscopic examination of organs and tissues did not reveal any treatment-related changes.

Based on the results of this study, viz. soft faeces (both sexes), decreased body weight gain (males), prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes) as observed in animals treated with the highest concentration of the test substance and decreased alkaline phosphatase activity as observed in males of the mid- and high-dose groups and in females of all dose-groups, the No Observed Effect Level (NOEL) for parental toxicity is <150 mg/kg body weight/day and the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 500 mg/kg body weight/day.