Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 15, 2016 to February 09, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD 407 and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is chosen as the test system because this species is commonly used for repeated dose oral toxicity testing and it meets the regulatory requirement of most of the regulatory agencies
Sex:
male/female
Details on test animals and environmental conditions:
The animal room was air-conditioned with adequate (above 10) air changes per hour.
The experimental room was continuously monitored for temperature and relative humidity.
The ranges for room temperature and relative humidity were 20.9°C to 23.2°C and 51 to 65%, respectively.
The animals were provided with a light cycle of 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral route is the intended route of administration in humans
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dose formulations were taken immediately after dilution of the concentrates with the diluent (vehicle) on treatment start date and treatment end date for homogeneity (mean of homogeneity is given as dose concentration) analyses. On week 3, samples of all dose formulations were analysed for dose concentrations by analysing triplicate samples. Analyses were performed by Analytical Chemistry division, RCC Laboratories India Private Limited according to an Analytical method provided by the sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
05
Control animals:
yes
Details on study design:
- Dose selection rationale: RCC Study No. 118
- All doses relate to the content of the active ingredient (trisodium trioxalatoferrate) and not to the reaction mass, which is the submission substance; e.g. 30 mg/kg bw/day correspond to 53 mg/kg bw/day (test substance actually administered) and 100 mg/kg bw/day correpond to 177 mg/kg bw/day (test substance actually administered).
- Post-exposure recovery period: 14 days

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Acclimatization Period (Once daily), Treatment Period ( Twice daily on first 3 days of treatment; once daily thereafter) and Recovery Period (Once daily)

BODY WEIGHT: Yes
- Time schedule for examinations: During Acclimatization and before randomization, Treatment & Recovery Periods (Once Weekly)

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Treatment & Recovery Periods (Once Weekly)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: : Once during acclimatization (In 20% of total population)
- Dose groups that were examined: Week 4 in all animals of groups 1 and 5

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 - Group 1 to 5 (Allocation A) and Week 6 - Group 1R and 5R (Allocation B)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 - Group 1 to 5 (Allocation A) and Week 6 - Group 1R and 5R (Allocation B)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 - Group 1 to 5 (Allocation A) and Week 6 - Group 1R and 5R (Allocation B)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of illness were observed in any of the male and female animals treated with test item at dose levels of 30, 100, 300 and 1000 mg/kg body weight throughout the experimental period
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in any of the male and female animals treated with test item at dose levels of 30, 100, 300 and 1000 mg/kg body weight during the treatment period of 28 days and recovery period of 14 days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain (%) of male and female animals of treatment and recovery groups were comparable with the control group animals. No significant changes were noted during the dosing period of 28 days and recovery period of 14 days
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the 28 consecutive days dosing period and post-dosing recovery period of 14 days the quantity of feed consumed by animals across different dose groups was found to be comparable with that of control animals. The statistically significant changes observed in the feed consumption are marginal and could not be attributed to the test item administration
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination did not reveal any abnormalities in the control and high dose treated animals at the end of treatment period
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters such as Erythrocyte count (RBC), Hemoglobin (Hb), Hematocrit (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Total leucocyte count (WBC), Differential leukocyte count (DC), Prothrombin time (PT) and Activated Partial Thromboplastin time (APTT) did not show any toxicologically relevant findings.
The statistically significant changes observed in the hematological parameters are marginal and could not be attributed to the test item administration as these values were within clinical and biological variation
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters like Glucose (GLU), Urea, Creatinine (CREA), Cholesterol Total (CHOL), Triglycerides (TRIGL), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Bilirubin (BIL), Sodium (Na), Potassium (K), Chloride (Cl), Total protein (TPO), Albumin (ALB), Globulin (GLB) and Albumin and Globulin ratio (A/G) did not show any toxicologically relevant findings in treatment and recovery groups.
The statistically significant changes observed in the clinical biochemistry parameters are marginal and could not be attributed to the test item administration as these values were within clinical and biological variation
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urine analysis parameters (volume, specific gravity, color, clarity, pH, Erythrocytes, Leukocytes, urobillinogen, bilirubin, ketone bodies, proteins, glucose and microscopic examination) did not reveal any test item related changes.
The statistically significant changes observed in the urine analysis parameters are marginal and could not be attributed to the test item administration as these values were within biological variation
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No significant test item-related differences were observed in organ weights.
The statistically significant changes observed in the organ weights were marginal and well within the control range and were considered to be biological variation
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy performed at the end of the treatment period revealed test item related red foci and/or thickened glandular stomach in male and female animals of high dose (1000 mg/kg) and intermediate-2 dose (300 mg/kg) groups. In addition, greenish black colored content in gastrointestinal tract was observed in male and female animals of high dose (1000 mg/kg), intermediate-2 (300 mg/kg) and intermediate-1 (100 mg/kg) groups. There was no abnormality observed in animals of low dose (30 mg/kg) and control groups. Following recovery period, the changes observed in gastrointestinal tract had returned to normal in high dose recovery (1000 mg/kg) group
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Under the conditions of this study, the test item - Sanodal Gold 4N trocken microscopically produced changes in stomach, kidneys and thymus. In high dose (1000 mg/kg) group, haemorrhages, apoptosis, mineralization and/or polymorph nuclear cells infiltrate were observed in glandular stomach and tubular dilatation with or without crystalline deposits in kidneys and apoptosis in thymus of male and female animals. In intermediate-2 (300 mg/kg) group, microscopically haemorrhages, apoptosis mineralization and/or polymorph nuclear cells infiltrate were observed in glandular stomach of male and female animals and tubular dilatation with crystalline deposits in kidneys only in female animals. In intermediate-1 (100 mg/kg) group, microscopically apoptosis was observed in glandular stomach of male animals and apoptosis or haemorrhages in glandular stomach in female animals. No abnormality was observed in low dose (30 mg/kg) group. The changes in glandular stomach were persistent after 14 day treatment free recovery period i.e. apoptosis and/or mineralization in male animals and apoptosis in female animals of high dose recovery (1000 mg/kg) group
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic effects
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
the value relates to trisodium trioxalatoferrate and not to the reaction mass
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Local effects
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
the value relates to trisodium trioxalatoferrate and not to the reaction mass
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

All determined effect levels relate to the active ingredient trisodium trioxalatoferrate and not to the reaction mass.

Applicant's summary and conclusion

Conclusions:
The test item was administered once daily to groups of Wistar rats for a period of 28 days at doses of 30, 100, 300 and 1000 mg/kg body weight (all doses relate to the active ingredient). No test item related changes in the body weights, feed consumption, haematology, clinical biochemistry, urine parameters and organ weights were noted.
Macroscopic examination revealed test item related red foci and/or thickened glandular stomach in male and female animals of high dose (1000 mg/kg) and intermediate-2 dose (300 mg/kg) groups. In addition, greenish black colored content in gastrointestinal tract was observed in male and female animals of high dose (1000 mg/kg), intermediate-2 (300 mg/kg) and intermediate-1 (100 mg/kg) groups. There was no abnormality observed in animals of low dose (30 mg/kg) and control groups. During recovery period, the changes observed in gastrointestinal tract subsided to normal in high dose recovery (1000 mg/kg) group.
Microscopic examination revealed haemorrhages, apoptosis, mineralization and/or polymorph nuclear cell infiltrate in stomach, tubular dilatation with or without crystalline deposits in kidneys and apoptosis in thymus attributable to the test item at 1000 mg/kg body weight (high dose). The apoptosis and/or mineralization in glandular stomach of male animals and apoptosis in glandular stomach of female animals of high dose recovery (1000 mg/kg) group were persistent but showed a tendency to recovery after 14 day treatment free recovery period.
In 300 mg/kg (intermediate-2) dose group, microscopic examination revealed haemorrhages, apoptosis, mineralization and/or polymorph nuclear cell infiltrate in stomach and tubular dilatation with or without crystalline deposits in kidneys. At 100 mg/kg (intermediate-1) dose group, haemorrhages and/or apoptosis was observed in stomach. No microscopic abnormality was observed at 30 mg/kg (low) dose group.
Hence, under the conditions of the experiment and based on the findings of the present study entitled “Repeated Dose 28 Days Oral Toxicity Study with the test item in Wistar Rats” the NOAEL (No Observed Adverse Effect Level) for Systemic effects was found to be the 100 mg/kg body weight and Local effects was found to be 30 mg/kg body weight
All determined effect levels relate to the active ingredient trisodium trioxalatoferrate and not to the reaction mass.
Executive summary:

Study Title: Repeated Dose 28 Days Oral Toxicity Study with the test item in Wistar Rats.

This study was conducted based on the OECD Guidelines for the Testing of Chemicals, Section 4, Health Effects, Number 407, 03rd October, 2008.

The purpose of this study was to assess the toxicity of the test item when administered once daily to Wistar rats by oral gavage for a period of 28 days. The reversibility of treatment related changes was assessed after a treatment free 14 day recovery period.

A non-GLP Dose Range Finding study (RCC Study No. 118) was carried out before the 28 days repeated dose study to confirm the high dose level for test item. A total of three groups (Low, Intermediate and High) consisting of 2 male and 2 female rats each were treated for a period of 14 days at dose levels of 100, 300 and 1000 mg/kg body weight (active ingredient) respectively. All animals were observed for mortality/viability, clinical signs of toxicity, feed consumption and body weight. At the end of treatment all animals were sent to necropsy for gross pathological and Histopathological examinations. Mild histopathological findings in low dose group were observed. So, an additional dose group was included in the 28 days repeated dose study. The doses for the 28 days repeated dose study are 30, 100, 300 and 1000 mg/kg body weight (active ingredient).

The test item was formulated in distilled water and administered once daily for 28 days to Wistar rats at dose levels of 30 (Low - G2), 100 (Intermediate 1 - G3), 300 (Intermediate 2 - G4) and 1000 (High - G5 & High Recovery - G5R) mg/kg body weight. Vehicle control 0 (G1 & G1R) mg/kg body weight was administered with distilled water for 28 days. Each group consisting of five males and five females were used for the main study and two groups each consisting of five males and five females were used for control recovery and high dose recovery respectively.

The following observations were performed: Mortality/viability, clinical signs, Ophthalmoscopy, body weights, feed consumption, clinical pathology (haematology, biochemistry and urinalysis), macroscopic and microscopic examination, organ weights and histopathology of the preserved tissues.  

Statistical analysis was performed using Stat plus software to compare the significant difference between treatment and control group of animals (t-test/ANOVA).  

Results

No mortality was observed in any of the male and female animals treated with test item at dose levels of 30, 100, 300 and 1000 mg/kg body weight (active ingredient) during the treatment period of 28 days and recovery period of 14 days.

No clinical signs of illness were observed in any of the male and female animals treated with test item at dose levels of 30, 100, 300 and 1000 mg/kg body weight (active ingredient) throughout the experimental period.

Ophthalmological examination did not reveal any abnormalities in the control and high dose treated animals at the end of treatment period.

During the 28 consecutive days dosing period and post-dosing recovery period of 14 days the quantity of feed consumed by animals across different dose groups was found to be comparable with that of control animals. The significant changes observed in the feed consumption are marginal and could not be attributed to the test item administration.

Body weight and body weight gain (%) of male and female animals of treatment and recovery groups were comparable with the control group animals. No significant changes were noted during the dosing period of 28 days and recovery period of 14 days.

Hematological parameters such as Erythrocyte count (RBC), Hemoglobin (Hb), Hematocrit (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Total leucocyte count (WBC), Differential leukocyte count (DC), Prothrombin time (PT) and Activated Partial Thromboplastin time (APTT) did not show any toxicologically relevant findings. The significant changes observed in the hematological parameters are marginal and could not be attributed to the test item administration as these values were within clinical and biological variation.    

Clinical biochemistry parameters like Glucose (GLU), Urea, Creatinine (CREA), Cholesterol Total (CHOL), Triglycerides (TRIGL), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Bilirubin (BIL), Sodium (Na), Potassium (K), Chloride (Cl), Total protein (TPO), Albumin (ALB), Globulin (GLB) and Albumin and Globulin ratio (A/G) did not show any toxicologically relevant findings in treatment and recovery groups. The significant changes observed in the clinical biochemistry parameters are marginal and could not be attributed to the test item administration as these values were within clinical and biological variation.

Urine analysis parameters (volume, specific gravity, color, clarity, pH, Erythrocytes, Leukocytes, urobillinogen, bilirubin, ketone bodies, proteins, glucose and microscopic examination) did not reveal any test item related changes. The significant changes observed in the urine analysis parameters are marginal and could not be attributed to the test item administration as these values were within biological variation.

No significant test item-related differences were observed in organ weights. The significant changes observed in the organ weights were marginal and well within the control range and were considered to be biological variation.

Necropsy performed at the end of the treatment period revealed test item related red foci and/or thickened glandular stomach in male and female animals of high dose (1000 mg/kg) and intermediate-2 dose (300 mg/kg) groups. In addition, greenish black colored content in gastrointestinal tract was observed in male and female animals of high dose (1000 mg/kg), intermediate-2 (300 mg/kg) and intermediate-1 (100 mg/kg) groups. There was no abnormality observed in animals of low dose (30 mg/kg) and control groups. Following recovery period, the changes observed in gastrointestinal tract had returned to normal in high dose recovery (1000 mg/kg) group.

Under the conditions of this study, the test item microscopically produced changes in stomach, kidneys and thymus. In high dose (1000 mg/kg) group, haemorrhages, apoptosis, mineralization and/or polymorph nuclear cells infiltrate were observed in glandular stomach and tubular dilatation with or without crystalline deposits in kidneys and apoptosis in thymus of male and female animals. In intermediate-2 (300 mg/kg) group, microscopically haemorrhages, apoptosis mineralization and/or polymorph nuclear cells infiltrate were observed in glandular stomach of male and female animals and tubular dilatation with crystalline deposits in kidneys only in female animals. In intermediate-1 (100 mg/kg) group, microscopically apoptosis was observed in glandular stomach of male animals and apoptosis or haemorrhages in glandular stomach in female animals. No abnormality was observed in low dose (30 mg/kg) group. The changes in glandular stomach were persistent after 14 day treatment free recovery period i.e. apoptosis and/or mineralization in male animals and apoptosis in female animals of high dose recovery (1000 mg/kg) group.

The analytical method was validated and the detector response was found to be linear (r2 = 1.000) in the range of 10 to 200 mg/L concentration.

The accuracy and precision obtained in the dose preparations were within recovery concentration (i.e. T1 - 98.39% and T2 - 98.32%).

The recovery content was found homogeneously distributed in the suspension at concentrations 3.0 mg/mL, 10 mg/mL, 30 mg/mL and 100 mg/mL for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 0.004 to 0.294).

The recovery content was obtained in all the dose groups were in agreement with the target concentration (i.e. 3.0 mg/mL – 95.77%, 10 mg/mL - 96.96%, 30 mg/mL – 96.05% and 100 mg/mL – 97.10%)

CONCLUSION

The test item was administered once daily to groups of Wistar rats for a period of 28 days at doses of 30, 100, 300 and 1000 mg/kg body weight (active ingredient). No test item related changes in the body weights, feed consumption, haematology, clinical biochemistry, urine parameters and organ weights were noted.

Macroscopic examination revealed test item related red foci and/or thickened glandular stomach in male and female animals of high dose (1000 mg/kg) and intermediate-2 dose (300 mg/kg) groups. In addition, greenish black colored content in gastrointestinal tract was observed in male and female animals of high dose (1000 mg/kg), intermediate-2 (300 mg/kg) and intermediate-1 (100 mg/kg) groups. There was no abnormality observed in animals of low dose (30 mg/kg) and control groups. During recovery period, the changes observed in gastrointestinal tract subsided to normal in high dose recovery (1000 mg/kg) group.

Microscopic examination revealed haemorrhages, apoptosis, mineralization and/or polymorph nuclear cell infiltrate in stomach, tubular dilatation with or without crystalline deposits in kidneys and apoptosis in thymus attributable to the test item  at 1000 mg/kg body weight (high dose). The apoptosis and/or mineralization in glandular stomach of male animals and apoptosis in glandular stomach of female animals of high dose recovery (1000 mg/kg) group were persistent but showed a tendency to recovery after 14 day treatment free recovery period.

In 300 mg/kg (intermediate-2) dose group, microscopic examination revealed haemorrhages, apoptosis, mineralization and/or polymorph nuclear cell infiltrate in stomach and tubular dilatation with or without crystalline deposits in kidneys. At 100 mg/kg (intermediate-1) dose group, haemorrhages and/or apoptosis was observed in stomach. No microscopic abnormality was observed at 30 mg/kg (low) dose group.

Hence, under the conditions of the experiment and based on the findings of the present study entitled “Repeated Dose 28 Days Oral Toxicity Study with the test item in Wistar Rats” the NOAEL (No Observed Adverse Effect Level) for Systemic effects was found to be the 100 mg/kg body weight and Local effects was found to be 30 mg/kg body weight. All effect levels relate to the active ingredient and not to the reaction mass.