Reaction mass of iron chelate of sodium salt N-[1,2 dicarboxyethyl] D,L aspartic acid and sodium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

1997-01-31 to 1997-03-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

Please see attached file.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Please see attached file.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

The feeding study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days.

GLP compliance:

not specified

Specific details on test material used for the study:

Sodium iron EDTA (FeEDTA; approx. 13% iron) and iron (II) sulfate (FeSO4; approx. 20% iron) were obtained from Dr Paul Lohmann (Emmerthal, Germany).

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rats were obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany.

Sex:

male

Details on test animals or test system and environmental conditions:

Two hundred and fifty young adult male rats [Sprague- Dawley (Crl:CD@BR)] were obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany. At the commencement of the treatment period, the age of the rats was 5-6 weeks, and the body weight variation did not exceed +/- 20% of the mean weight. The rats were housed indi- vidually under conventional laboratory conditions in one room, in suspended stainless-steel cages fitted with wire-mesh floor and front.
After arrival, the rats were acclimatized to the laboratory conditions and maintained on the AIN-93G diet for at least 14 days prior to the start of the experiment. On the experimental start date the animals were allo- cated to the various groups by computer randomization.

Route of administration:

oral: feed

Details on route of administration:

oral administration via the diet for 31 and 61 days

Vehicle:

other: AIN-93G diet

Details on oral exposure:

Prior to the start of the experiment, a batch of modified AIN-93G (Reeves et al., 1993) diet was prepared freshly at TNO and stored at or below18oCuntil use. Two batches of a commercially available low-iron AIN-93M diet (<5 mg iron/kg diet; lot nos 8315-6 and 8345-4) were obtained from Dyets Inc. (Bethlehem, PA, USA). FeEDTA or FeSO4 were incorporated in the low-iron AIN-93M diet at constant concentrations by mixing in mechanical mixers at TNO. The composition of the diets are given below:

Composition of the modified AIN-93 diets

AIN-93G diet(a) Low iron AIN-93G diet(b)
Ingredients (%) (%)
Casein 20.00 14.00
L-Cystine 0.30 0.18
Wheat starch 62.95
Corn starch 46.5692
Dyetrose(c) 15.50
Sucrose 10.00
Cellulose 5.00 5.00
Choline bitartrate 0.25 0.25
AIN-93G mineral mixture 3.50
AIN-93M mineral mixture(d) 3.50
AIN-93VX vitamin mixture 1.00 1.00
Soybean oil 7.00
Soybean oil (stabilized with TBHQ) 4.0008
Total(e) 100.00 100.00

a Diet prepared by TNO.
b Diet obtained from Dyets Inc., Bethlehem, PA, USA.
c Dyetrose: dextrinized corn starch containing 90-94% tetra- saccharides (Reeves et al., 1993).
d AIN-93M mineral mixture without iron.
e FeSO4 or FeEDTA were mixed with these diets to obtain diets containing 35, 70 or 140 mg Fe/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content, stability and homogeneous distribution of iron in the test diets was analyzed by means of atomic absorption spectrometry (AAS). The content of each of the six diets was measured three times during the study.

Duration of treatment / exposure:

31 or 61 days

Frequency of treatment:

ad libitum

Dose / conc.:

35 mg/kg diet

Remarks:

Fe from FeSO4, low dose

Dose / conc.:

35 mg/kg diet

Remarks:

fe from FeEDTA, low dose

Dose / conc.:

70 mg/kg diet

Remarks:

fe from FeSO4, mid-dose

Dose / conc.:

70 mg/kg diet

Remarks:

Fe from FeEDTA mid-dose

Dose / conc.:

140 mg/kg diet

Remarks:

Fe from FeSO4, high-dose

Dose / conc.:

140 mg/kg diet

Remarks:

Fe from FeEDTA, hig-dose

No. of animals per sex per dose:

40 male rats per dose

Control animals:

yes, concurrent no treatment

Details on study design:

The experiment comprised six main groups: three test groups receiving different levels of FeEDTA and three control groups, receiving different levels of FeSO4. Each group consisted of 40 males. Twenty rats of each group were sacrificed after 31 days of feeding and 20 rats of each group were sacrificed after 61 days of feeding. In addition, a group of 10 untreated male rats was sacrificed at the start of the experiment, in order to establish baseline levels of the analytical parameters to be mea- sured. No other data were collected for these animals.

Positive control:

No

Observations and examinations performed and frequency:

Each animal was observed daily and all abnormal- ities, signs of ill-health or reactions to treatment were recorded.

Body weights
The body weight of each animal was recorded once during the acclimatization period, at initiation of treat- ment, and once weekly thereafter. In addition, the animals were weighed on the day of scheduled autopsy in order to determine their correct organ to body weight ratios.

Food consumption
Food consumption was measured per animal twice weekly, over periods of 3 or 4 days, by weighing the feeders. The results were expressed in g per animal per day. The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.

Intake of iron from FeEDTA or FeSO4
The intake of iron from FeEDTA or FeSO4 per kg body weight per day was calculated from the nominal dietary level of iron, the food intake, and the body weight (mean of the body weights measured at the start and the end of each week).

Sacrifice and pathology:

Gross necropsy
After completion of the treatment periods, the animals were killed on several successive working days, in such a seąuence that the average time of killing was approximately the same for each group. The animals were killed by ex- sanguination from the abdominal aorta under ether anes- thesia and then examined macroscopically for pathological changes. The adrenals, brain, caecum, colon, heart, kidneys, liver, oesophagus, rectum, small intestines (duodenum, ileum, jejunum), spleen, stomach, testes and thymus, were excised, examined for gross lesions and preserved in a neutral aąueous phosphate buffered 4% solution of formaldehyde (10% solution of formalin). The organs that are italicised were weighed prior to preservation.

Histopathological examination
Samples of liver, spleen and all gross lesions were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin and with Perl's Prussian blue for iron. The stained sections, except the Perl's Prussian blue stained sections of gross lesions, were examined by light microscopy by an experienced pathologist.

Non-heme iron analysis in tissue
Fresh samples of the liver, spleen and kidneys of all animals were analyzed for non-heme iron content by the bathophenanthroline reaction and the results were expressed as µg Fe/g tissue (wet weight). The analysis was performed according to the method described by Whittaker et al. (1997). Before analysis of study samples, the method was validated for the different tissues under investigation.

Other examinations:

Hematology
Approximately 16 h before the rats were scheduled for autopsy, they were deprived of food. At autopsy, blood samples were taken from the abdominal aorta of the rats, while under ether anesthesia. K2-EDTA was used as anticoagulant. In each sample the following determi- nations were carried out: hemoglobin, packed cell volume, red blood cell count, total white blood cell count, differential white blood cell count, prothrombin time and thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean cor- puscular hemoglobin concentration (MCHC).

Clinical chemistry
Approximately 16 h before the rats were scheduled for autopsy, they were deprived of food. At autopsy, blood samples were taken from the abdominal aorta of the rats, while under ether anesthesia, in heparinized plastic tubes and plasma was prepared by centrifugation. The fol- lowing measurements were made in the plasma using a Hitachi 911 analyzer: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), ala- nine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate.


Determination of iron and total iron binding capacity (TIBC) in blood plasma
Approximately 16 h before the rats were scheduled for autopsy, they were deprived of food. At autopsy, blood samples were taken from the abdominal aorta of the rats, while under ether anesthesia, in heparinized plastic tubes and plasma was prepared by centrifugation. The plasma iron concentration was determined with Ferro- zine without deproteinization in each sample, using Boeringer kit no. 1533704 in a Hitachi 911 analyzer. The TIBC was determined in all samples according to Boeringer kit no. 759422 in a Hitachi 911 analyzer. The principle of this test is as follows: the plasma is treated with an excess of Fe3+ ions to saturate the transferrin. Uncomplexed iron is precipitated with basic magnesium carbonate and the iron in the supernatant is determined.

Statistics:

Body weights were analyses by one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests, food consump- tion and food conversion efficiency were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, plasma iron, TIBC and organ weights were analyzed by one-way ANOVA followed by Dunnett's multiple comparison tests, relative differential white blood cell counts were analyzed by Kruskal-Wallis non- parametric ANOVA followed by Mann-Whitney U-tests, histopathological changes were analyzed by Fisher's exact probability test and non-heme iron analyses in tissue were analyzed by two-way ANOVA followed by Student's t-tests. All tests were two-sided. Probability values of P<0.05 were considered significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical signs observed during the experiment included encrustations around the eyes, malocclusion of incisors, nasal encrustations, red discharge from the nose, sniffing, sparsely haired skin, dermal encrustations and dermal wounds.
The signs that were observed are common findings in Sprague-Dawley rats and were randomly distributed among the various groups. These observations were not considered to be related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences in mean body weights were observed among the groups throughout the experiment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No remarkable differences in overall mean food con- sumption were observed during the experiment. Occasion- ally, at some time points during the study, statistically significant differences in food consumption were seen, but these were not related to the treatment and were considered chance findings. The overall mean food consumption values for the six groups ranged from 23.6 to 24.5 g/rat/day. FCE figures showed a number of statistically sig- nificant differences among the groups in weeks 1, 3 and 4. In most of these cases the FCE was significantly lower in the high-dose FeSO4 group. In weeks 2 and 5-8, no statistically significant differences in FCE were observed (data not shown).

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

The daily intake of iron per kg body weight from FeEDTA and FeSO4 decreased in the course of the study, due to the well-known decrease in food con- sumption per kg body weight with increasing age of the rats. The mean intakes of iron in the low-dose FeSO4, the low-dose FeEDTA, the mid-dose FeSO4, the mid- dose FeEDTA, the high-dose FeSO4 and the high-dose FeEDTA groups were 2.84, 2.81, 5.69, 5.67, 11.54 and 11.19 mg/kg body weight/day, respectively.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 31 days of feeding, the mean corpuscular hemo- globin was statistically significantly higher in groups receiving the high dose of FeSO4 and the mid and high dose of FeEDTA in comparison to the group receiving the low dose of FeSO4. The mean corpuscular hemoglobin concentration was statistically significantly higher in the group receiving the high dose of FeEDTA in comparison to the group receiving the low dose of FeSO4. No other statis- tically significant differences in red blood cell or coagula- tion variables were observed after 31 days of feeding. After 61 days of feeding, no statistically significant differences in red blood cell or coagulation variables were observed.
After 31 days of feeding, the absolute number of eosinophils was statistically significantly higher in the high-dose FeEDTA group in comparison to the low- and mid-dose FeEDTA groups and the mid-dose FeSO4 group. The percentage of eosinophils was statistically significantly higher in the high-dose FeEDTA group in comparison to the low-dose FeEDTA and the mid-dose FeSO4 groups. No other statistically significant differ- ences in white blood cell counts were observed.
After 61 days of feeding, no statistically significant differences in white blood cell counts were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At day 32
Alkaline phosphatase activity was decreased in the mid-dose FeEDTA group in comparison to the low-, mid- and high-dose FeSO4-groups. At day 62, this dif- ference was no longer present. The concentration of total bilirubin was higher in the mid-dose FeSO4 group in comparison to the low-dose FeEDTA and FeSO4- groups. At day 62, this difference was no longer present. Sodium and chloride concentrations decreased with increasing dietary levels of iron (from both sources). At day 62, the difference in chloride concentrations was no longer present.

At day 62
Total protein concentrations were lower in the FeEDTA groups than in the FeSO4 groups and albumin concentrations were lower in the mid- and high-dose FeEDTA groups than in the low-dose FeEDTA group and the low- and mid-dose FeSO4 groups. The calcium concentration was lower in the high-dose FeEDTA group than in the low-dose FeSO4 and FeEDTA groups and the mid-dose FeSO4 group. Sodium concentrations were lower in the high-dose FeEDTA and FeSO4 groups than in the low-dose FeSO4 group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences in terminal body weights and absolute and relative organ weights were observed among the groups after 31 days of feeding. After 61 days of feeding, the absolute and relative thymus weights in the low-dose FeEDTA group was statistically significantly higher than those in the low-dose FeSO4 and the mid- and high-dose FeEDTA groups. No other significant differences in terminal body weights and absolute and relative organ weights were observed among the groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination did not reveal treatment- related changes. The changes observed are considered common for rats of this strain and age, with the possible exception of a nodule in the abdominal cavity in a high-dose FeEDTA animal sacrificed after 61 days of feeding. Moreover, they occurred in one or a few animals only or they were distributed about eąually among the groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination did not reveal histopatho- logical changes that could be related to either the dose or the type of iron. Fat necrosis was the diag- nosis of the macroscopically observed nodule in the abdominal cavity, a lesion which has been occasionally observed in historical controls and was therefore con- sidered to be a fortuitous finding, unrelated to the treatment. The other changes observed are considered unremarkable. Moreover, they occurred in one or a few animals only or were distributed about eąually among the groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of Prussian blue-stained sections

After 31 days of feeding, no positive Prussian blue reaction was observed in the liver. After 61 days of feeding, a positive Prussian blue reaction in the liver was observed in 11, nine and nine animals of the low-, mid- and high-dose FeEDTA groups, respectively, and in six, 15 and 14 animals of to the low-, mid- and high-dose FeSO4 groups, respectively. In animals with a positive reaction, the blue staining was found predominantly in the Kupffer cells. The incidences of blue staining in hepatocytes and Kupffer cells of livers of the FeEDTA- exposed animals was not influenced by the dose. In the mid- and high-dose FeSO4 groups, the incidences of blue staining in the Kupffer cells were higher than those in the low-dose FeSO4 group.
After 31 days of feeding, the spleen of all animals, low
Fe-group animals included, demonstrated a positive Prussian blue reaction. Blue staining was mainly con- fined to the red pulp, and the degree (intensity of the staining and the number of positive cells) varied from very slight to slight. The incidence of slight blue staining tended to be somewhat higher in the high-dose FeSO4 and FeEDTA groups compared to the low Fe-groups. No differences were observed between the FeSO4 and FeEDTA groups maintained on diets with comparable iron content.
After 61 days of feeding, a positive reaction in the spleen was observed in all animals. Compared to animals exposed for 31 days, the degree of staining in the spleen of animals exposed for 61 days was somewhat increased, and the staining was extended from red pulp only to red pulp and marginal sinus, especially in animals with moderate blue staining. A distinct shift from (very) slight to moderate degree of blue staining was observed in the high-dose FeSO4 group when compared to the low-dose FeSO4 group, and in the mid- and high-dose FeEDTA groups when compared to the low-dose FeEDTA group.

Details on results:

In the present study, the dietary exposure to iron from FeSO4 or FeEDTA did not result in toxicologically relevant changes in clinical signs, body or organ weights, food consumption, red and white blood cell or coagulation variables or in pathology. The clinical chemistry examination revealed one change that may be related to treatment. The observed lower plasma cal- cium concentration after 61 days of feeding FeEDTA may be related to the chelating properties of EDTA. Calcium is (partly) bound to plasma proteins, and total calcium is greatly influenced by protein concentrations, mainly albumin. Concurrent with the decrease in cal- cium, the plasma concentration of albumin was decreased with approximately 2 g/l. It has been esti- mated that 1 g of albumin is capable of binding 0.02 mmol of calcium (Endres and Rude, 1999). Therefore, the decrease of 2 g/l of albumin may be responsible for a decrease of 0.04 mmol/l of calcium. Under this assumption, the lower calcium concentration in the high-dose FeEDTA group is for the larger part explained by the lower albumin levels and not by binding to EDTA. The most common causes for decreased plasma albumin levels are inflammatory processes and hepatic disease (Johnson et al., 1999). The activities of ALAT, ASAT and ALP (marker enzymes for liver damage) were not influenced by the treatment, indicating that hepatic damage was absent. Moreover, macro- scopic and microscopic examination did not provide evidence of liver damage or any inflammatory processes. Therefore, the lower calcium concentration observed in the clinical chemistry examination in plasma is not considered to be of toxicological significance.

Key result

Dose descriptor:

NOAEL

Remarks:

for FeSO4

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

for Fe from FeEDTA

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Conclusions:

It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO4. Moreover, feeding iron up to 11.5 and
11.2 mg/kg body weight/day, derived from FeSO4 and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects.

Executive summary:

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate. It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than ironfromFeSO4.Moreover,feedingironupto11.5and11.2mg/kgbodyweight/day,derivedfromFeSO4andFeEDTA,respec-

tively, did not result in tissue iron excess nor in any other toxicologically significant effects.©2001 Elsevier Science Ltd. All rights

reserved.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

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:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

144538-83-0

Cas Number:

144538-83-0

IUPAC Name:

144538-83-0

Constituent 2

Reference substance name:

2-(1,2-Dicarboxy-ethylamino)-succinic-acid tetra- Na-salt

IUPAC Name:

2-(1,2-Dicarboxy-ethylamino)-succinic-acid tetra- Na-salt

Constituent 3

Reference substance name:

Imminodisuccinic acid, tetrasodium salt

IUPAC Name:

Imminodisuccinic acid, tetrasodium salt

Details on test material:

- Name of test material (as cited in study report): Iminodisuccinat, Na-Salz
- Molecular formula (if other than submission substance): C8H7NNa408
- Molecular weight (if other than submission substance): 337 g/mol
- Smiles notation (if other than submission substance): C(C(C(=O)[O-])NC(CC(=O)[O-])C(=O)[O-])C(=O)[O-].[Na+].[Na+].[Na+].[Na+]
- InChl (if other than submission substance): InChI=1S/C8H11NO8.4Na/c10-5(11)1-3(7(14)15)9-4(8(16)17)2-6(12)13;;;;/h3-4,9H,1-2H2,(H,10,11)(H,12,13)(H,14,15)(H,16,17);;;;/q;4*+1/p-4
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: chelating agent
- Physical state: powder (white)
- Lot/batch No.: SAV В 0004
- Stability under test conditions: formulations were stable over the period of 8 days.
- Storage condition of test material: room temperature
- Other:

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
strain Hsd Cpb:WU
- Source: Harlan-Winkelmann, formerly Winkelmann, Borchen
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: Males: 117 (102 - 130) g; Females: 106 (89- 120) g
- Fasting period before study: not reported
- Housing: During acclimation and experimental period animals were kept individually
- Diet (e.g. ad libitum): ad libitum (Altromin® 1324 pellets)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24.01.1997 (delivery date) To: 28.02.1997 (Necropsy (End of Treatment) and 14.03.1997 Necropsy (End of Recovery Period).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test compound was mixed daily with the selected vehicle at the appropriate concentrations at room temperature;
- Storage of Formulation(s): at room temperature;
- Formulations were stable over a 8 days.

VEHICLE

- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytic investigations of the idenitity of the test compound and material balance were carried out and showed the content of Iminodisuccinat, Na-Salz to be 67.2%. Due to time-pressure the study was started before the result of the analytical report on material balance was available. For the calculation of the dosages the result of a preliminary analytical examination was used. In this preliminary examination the content was described to be 71% (declared by the Sponsor Jan. 28, 1997).
The calculation of the amount of test substance to be administered daily was done taking into account a content of 71%. The amount of test substance per dose was 100 % calculated on the actual basis of the content on 71%.
Analytic investigations on homogeneity and stability of the test substance in the administration vehicle covering the concentration range used were initiated before start of this study. As the result of this analytical investigations were available not before the start of the study the test substance was formulated daily. The investigations demonstrated homogeneity and stability in the administration medium over the period of 8 days.

Duration of treatment / exposure:

4 weeks + 2 weeks recovery period

Frequency of treatment:

daily (7 days/week)

Doses / concentrations

Remarks:

Doses / Concentrations:
38, 189 and 947 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 dosage scheme for the present study was based on the following investigations with Iminodisuccinat, Na-Salz:
The acute oral toxicity in rats is low with an LD50 oral > 2000 mg/kg body weight (study no. T 4060981; Study report 25566). No signs of toxicity were observed after single oral application of 2000 mg/kg body weight.
In addition to this, a range-finding study was conducted in which 1000 mg/kg body weight was administered to 3 male and 3 female Wistar rats for 6 days. No signs of toxicity were observed and the section gave no evidence of pathological changes.
On the basis of these results the following dose scheme was selected for the present subacute study:
0 (vehicle control) - 40 - 200 -1000 mg/kg body weight/day.
The actual administered dose was calculated taking into account a content of 71%. The analytical material balance (February 27, 1997) showed the actual content to be 67.2%, therefore the target dose was not fully reached and the actual dose administered was:
0 (vehicle control) - 38 -189 - 947 mg/kg body weight/day.
Because of the only minor deviation of the actual dose by comparison with the target dose, the dosages given in this report are generally the target doses.

- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily at two single days, on weekends and public holidays


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
At week 0, 1, 2 and 3 (main groups and recovery groups) and week 5 and 6 (recovery groups) a detailed clinical observation outside the homecage and during handling was performed using some elements of the functional observational battery (FOB) in all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
Food intake was calculated for all animals per group individually once a week from the difference of food supplied and not consumed. From these primary data the following were calculated for each week:
a) daily food intake per animal and kg body weight
b) mean daily food intake per animal and kg body weight.
For the total feeding period (for recovery groups calculations were done separatedly for treatment and recovery period):
c) mean food intake per animal kg body weight and day
d) mean food intake per animal per day
The calculation of the cumulative data was based on the 28- and 42-day exposure period(s) for main and recovery groups, respectively:
e) cumulative food intake per animal
f) cumulative food intake per kg body weight and day

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the week(s) 4 (main groups) and 6 (recovery groups)
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: No
- How many animals: all animals per group in the week(s) 4 (main groups) and 6 (recovery groups)
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the week(s) 4 (main groups) and 6 (recovery groups)
- Animals fasted: No
- How many animals: all animals per group in the week(s) 4 (main groups) and 6 (recovery groups)
- Parameters checked in table [No. 1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Motor activity: All rats were tested between 8:00 and 12:00 a.m. for 2 consecutive days during one time point of the study: week 4. At the first day all animals in the main groups were tested. At the second day all animals of the recovery groups were tested.
- At week 4 a functional observational battery (FOB) was conducted including handling of the animals, cage-side and open field observations as well as reflex/physiologic observations and measurements, landing foot splay and body temperature within 2 days in all animals. The FOB of tests was conducted with each animal tested individually. Scoring criteria and explicitly defined scales were used to rank the severity of observations that can not readily be quantified. The procedures used to determine landing foot splay and grip strength are based on established methods (cited in study report: Edwards and Parker, 1977; Meyer et al. 1979).
- Dose groups that were examined: Motor activity (all dose groups); FOB (at the week 4)
- Battery of functions tested: motor activity, handling of the animals, cage-side and open field observations as well as reflex/physiologic observations and measurements, landing foot splay and body temperature.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
At the end of the treatment period (main groups) or at the end of the recovery period (recovery groups) all surviving animals were necropsied. The were killed by exsanguination under diethyl ether anesthesia. The necropsy was a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. Changes were described in terms of localization, size, color and consistency whenever appropriate.

HISTOPATHOLOGY: Yes
Liver, heart, kidneys, lungs, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, peyer's patches, aorta, urinary bladder, pancreas, thymus, lymph nodes, spleen, salivary glands, lacrimal glands, Harderian glands, brain, spinal cord, sciatic nerve, trachea, esophagus, testes, epididymides, prostate, seminal versicles, ovaries, oviducts, uterus, vagina, pituary gland, thyroid gland, parathyroid gland, adrenal glands, femur, sternum, tongue, sceletal muscle, skin and eyes were examined in the vehicle control group and the high dose groups (main groups). Liver, heart, kidneys and lungs were examined in addition in the low dose and mid dose group animals. The thymus was examined in the recovery groups.

Other examinations:

The following organs of the animals killed at the end of the treatment and the recovery period were weighed before fixation:
brain, heart, liver, spleen, kidneys (both), adrenal glands (both), ovaries and testes (both), epididymides, thymus.

Statistics:

The statistical evaluation of data related to clinical chemistry, hematology, body and organ weights as well as feed and water intake is performed using SAS® routines. The data related to motor activity and functional observation battery (FOB) were not statistically evaluated as such an evaluation is considered to be of low relevance with groups of 5 animals and a wide individual variability of the parameters. The calculation of the standard deviations of some parameters of FOB was based on the rounded mean values of each animal.
Statistical evaluations on body weight and organ weight data were done using the Dunnet-test in connection with a variance analysis. A Kruskal-Wallis-Test with a Steel-Test was performed when data of feed intake were analyzed. Further statistical tests used to evaluate the remaining parameters are described in details in the study report.
- significant differences from the control group are indicated with "+" for p < 0.05 and "++" for p < 0.01.

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

Description (incidence and severity):

statistically significant decrease without toxicological relevance

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

no toxicological relevant deviations from control values

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

some single significantly different values without toxicological relevance

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

decreased motor activity in males in the highest dose group; no treatment related effects in FOB

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

significantly reduced absolute thymus weight in females in the recovery group; reduced relative thymus weights in the male and female animals in the recovery group. These effects considered to be without toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

no treatment related findings

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no signs of toxicity.
There was no evidence of a substance-related effect on mortality.

BODY WEIGHT AND WEIGHT GAIN
Growth of male and female rats was not significantly affected by treatment with the test substance.

FOOD CONSUMPTION
No toxicologically relevant differences in mean food consumption per kg body weight/day were detected in both sexes in all main groups up to week 4 and in the recovery groups up to week 6. The statistically significant decrease of food consumption in the females in the 1000 mg/kg recovery group at week 4 (TS 1%) or week 2, 3, 4 and 6 (TS 5%) is not regarded to be of toxicological relevance as such an effect was not seen in the main groups in females and all groups in males.

HAEMATOLOGY
There were no toxicological relevant deviations from control values. Some single values marked by deviating in a statistically significant manner in the females are not considered to be of toxicological relevance, as the differences from the control and other dose groups are minimal and/or no relation to dose or time was evident. The examinations of these parameters at the end of the recovery period revealed values comparable to controls.

CLINICAL CHEMISTRY
The test substance had no noticeable effect on the plasma enzymes and substrates investigated. Some single values marked by deviating in a statistically significant manner are not considered to be of toxicological relevance, as the differences from the control and other dose groups are minimal and/or no relation to dose or time was evident. The examinations of these parameters at the end of the recovery period revealed values comparable to controls.
The determination of blood electrolyte concentrations did not indicate treatment-related effects.
Occasionally, one mean of electrolyte concentrations (Na) was identified as being significantly different from controls in the 40 mg/kg dose group in males. However, this difference is of no toxicological relevance since is was not distributed dose dependently and the deviation was only minimal. The examinations of the electrolytes at the end of the recovery period revealed values comparable to controls.


NEUROBEHAVIOUR
Motor activity:
The horizontal activity was measured with 4 samples (10 minutes/sample) in each animal. There was a wide variety of individual scores for the horizontal activity which complicate the interpretation of this measurements. Taking into account the group means for each sample there was no effect on the motor activity up to 200 mg/kg body weight in males and up to 1000 mg/kg body weight in females. With 1000 mg/kg body weight in the males the motor activity at the end of the total sampling period (sample 4) was below that of the respective control in the main group and in the recovery group (see Table 3).
At week 4 a functional observational battery (FOB) was conducted including handling of the animals, cage-side and open field observations as well as reflex/physiologic observations and measurements, landing foot splay and body temperature within 2 days in all animals.
At week 0, 1, 2, 3 (main groups and recovery groups) and week 5 and 6 (recovery group) a detailed clinical observation outside the homecage and during handling was performed using some elements of the functional observational battery (FOB) in all animals.

No indication of treatment related effects were recorded during the study at all observed parameter in week 4. A trend to a decreased body temperature in the males in the 1000 mg/kg main group was not confirmed by the data generated in the same week in the males in the 1000 mg/kg recovery group and is therefore not considered to be treatment related. The detailed clinical observations with some elements of the functional observational battery also did not show any treatment related effects.

ORGAN WEIGHTS
There was no treatment related significant effect on the absolute weight of brain, heart, liver, spleen, testes/ovaries, epididymis, kidneys, adrenals and thymus in the males and females in the main groups as well as in the males in the recovery groups. The mean absolute thymus weight was significantly lower in females in the 1000 mg/kg recovery group than in the respective control group. The relative organ weight of brain, heart, liver, spleen, testes/ovaries, epididymis, kidneys, adrenals and thymus were not significantly affected in all other main groups. In the recovery groups there were lower mean relative thymus weights in the male and female animals. In all dose groups there was a high individual variability of relative and absolute thymus weights.

GROSS PATHOLOGY
At the end of the treatment period gross pathological examinations revealed no treatment related findings. All findings observed during necropsy occured in few animals only. These findings are known from control animals of previous studies in rats of that age. Thus, they are considered to be of spontaneous origin.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination of main group animals revealed no evidence of toxic effects in the treated animals (up to 1000 mg/kg body weight). All findings listed in the histopathology finding tables are distributed throuout all dose groups and/or known from control animals of previous studies in rats of that age. Thus, they are considered to be of spontaneous origin.

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

1

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: based on reduced motor activity in the highest dose group (main and recovery)

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no toxicologically relevant findings in all dose groups (main and recovery).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analyses of the test substance in the administration vehicle

The analytical investigation of homogeneity and stability of Iminodisuccinat, Na-Salz in the administration vehicle was started before the study start. These analytical investigations showed the test substance to be homogeneously distributed and stable in the concentration range used beyond the period of use (67.2 %). The content of the test substance in the administration vehicle was checked at least twice during the study. The data of the first check (February 14, 1997) showed that the test compound content agreed with the target concentrations for the mid- and high-dose-formulation, but the content was too low in the low-dose-formulation. The second check (February 25, 1997) verified that the test compound content agreed with the target concentrations within the defined limits.

Table 3. Motor activity - Horizontal activity counts in main and recovery groups (week 4)

	male	female	male (rec)	female (rec)
Control
sample1	2426	2818	3234	4094
sample 2	1781	1541	2448	2395
sample 3	1454	1364	1847	2260
sample 4	1133	950	974	777
40 mg/kg
sample 1	2372	2682
sample 2	1567	1706
sample 3	1133	1100
sample 4	952	1031
200 mg/kg
sample 1	2513	2557
sample 2	1932	1508
sample 3	1360	1417
sample 4	1112	1178
1000 mg/kg
sample 1	2173	2012	3379	3435
sample 2	1218	1146	1767	1997
sample 3	930	632	1058	1420
sample 4 '	513	830	482	947

sample = 10 min

Applicant's summary and conclusion

Conclusions:

Under the conditions described the administration of Iminodisuccinat, Na-Salz to male and female rats was tolerated without treatment-related lesions up to and including 200 mg/kg body weight in" the males and up to and including 1000 mg/kg body weight in the females. Therefore the no observed adverse effect level (NOEL) for the daily administration of Iminodisuccinat, Na-Salz is considered to be 200 mg/kg body weight in males and 1000 mg/kg body weight in females. The toxicological relevance of the isolated effect on motor activity in the males with 1000 mg/kg body weight is questionable.

Executive summary:

Iminodisuccinat, Na-Salz was administered orally to Wistar rats (5 males and 5 females per dose) once a day, by gavage in target doses of 0 (vehicle control) - 40 -200 - 1000 mg/kg body weight over a period of 4 weeks. In addition, 5 male and 5 female rats per group were treated with the vehicle or 1000 mg/kg body weight and observed for reversibility, continuance or delayed occurence of toxic effects during a recovery period of 14 days.

Mortality was unaffected by treatment with Iminodisuccinat, Na-Salz.

Appearance, clinical findings and general behaviour were not altered by treatment with the test substance up to and including 1000 mg/kg body weight.

Growth and food consumption were not affected by the treatment.

Hematological investigations gave no indication of toxicologically relevant damage to blood, hematopoetic organs or coagulability up to and including 1000 mg/kg body weight.

Clinical laboratory tests produced no evidence of treatment-related metabolic or organ damage.

Gross and histopathological investigations of various organs and tissues gave no indication of test-compound-related functional or morphological changes in both sexes up to and including 1000 mg/kg body weight. The organ weights were unaffected with the exception of a decrease of relative thymus weight in the recovery group (1000 mg/kg body weight). Taking into account the discrepancy of observations in the main groups and in the recovery groups and the fact that there was no evidence of treatment related effect in haematology examinations as well as in the histopathological examinations of thymus in the main group and the recovery group and of adrenals, spleen, draining and distant lymph nodes and bones in the main groups the decrease of relative thymus weight is considered to be Of no toxicotogical relevance.

The assessment of motor activity (horizontal activity) showed a wide variety of individual values. There was no effect on the motor activity up to 200 mg/kg body weight in both sexes. With 1000 mg/kg body weight the motor activity in the males in the main groups and in the recovery groups was below that of the respective control group at the end of the sampling period. Functional observations as another marker for neurotoxicity gave no evidence of a neurotoxic potential. In addition, the histopathological examination of brain, spinal cord and sciatic nerve gave no evidence of a neurotoxic action of the test material. In conclusion, taking into account all available data with regard to neurotoxicity, the toxicological relevance of the findings with regard to motor activity is questionable.

Under the conditions described the administration of Iminodisuccinat, Na-Salz to male and female rats was tolerated without treatment-related lesions up to and including 200 mg/kg body weight in" the males and up to and including 1000 mg/kg body weight in the females. Therefore the no observed adverse effect level (NOEL) for the daily administration of Iminodisuccinat, Na-Salz is considered to be 200 mg/kg body weight in males and 1000 mg/kg body weight in females. The toxicological relevance of the isolated effect on motor activity in the males with 1000 mg/kg body weight is questionable.