Registration Dossier

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:
1997-01-31 to 1997-03-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
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 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

open allclose all
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)
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.