Registration Dossier

Administrative data

Description of key information

oral:

- NOAEL (subacute, rat, m/f) = 250 mg/kg bw/day (OECD 407)

- NOAEL (subchronic, rat, m/f) = 103 mg/kg bw/day (OECD 408)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Oct 2012 - 24 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 03 October 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain specification: Crl: WI(Han)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 152 - 172 g (males), 130-150 g (females)
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 190612)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1039), ad libitum
- Water: tap water (sulphur acidified to a pH of approximately 2.8), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation was recorded for all dosing formulations. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. Before the first administration, the pH value of the formulations was measured.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 15, 50 and 120 mg/mL (low-, mid- and high dose)
- Amount of vehicle: 5 mL/kg bw
- Manufacturer: Fresenius Kabi (Ampuwa)
- Lot/batch no.: 19F630WA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the concentration of test item in dosing formulations, samples of at least 25 mL were retained from all groups once weekly during the treatment period and stored between -15 and -35 °C. Stability of the dosing formulations was tested once at the beginning of the treatment period. From the low and high dose groups samples of dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C. In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high and low dose formulations and stored between -15 and -35 °C.
The determination of test item concentration in the dosing formulations was performed by Eurofins Agrosciences Services EcoChem GmbH. The analytical method met the requirements of guideline SANCO/3029/99. Formulation analytics were performed as stand-alone study. Analytical results were provided to the study director for calculation of the actual test item concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- 10 per sex in the control and high dose groups
- 5 per sex in the low and mid dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the previous 14-day range-finding study (section 7.5.1: supporting, Allingham, 2013, 14-day range finder, rat, RL2)
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day
- Cage side observations included: health condition (once daily), morbidity and mortality (twice daily, except on weekends and public holidays when observations were made once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first administration and at least once a week thereafter
- Detailed clinical observations included observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period

FOOD CONSUMPTION:
- Time schedule for examinations: weekly during the treatment and recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes (overnight)
- Parameters examined: colour/ appearance , specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period
- Dose groups that were examined: all dose groups
- Battery of functions tested: multiple detailed behavioural observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
ORGAN WEIGHTS: Yes (all surviving animals: liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart; paired organs were weighed separately)
HISTOPATHOLOGY: Yes (all animals of the control and high dose groups: brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females), liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), small and large intestines (including Peyer´s patches), prostate and seminal vesicles with coagulating glands as a whole (males), stomach, urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid glands, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, sternum with bone marrow, lung and trachea, pituitary gland, mammary glands, oesophagus, skin, gross lesions; kidneys, urinary bladder and stomach (glandular and non-glandular) and gross lesions were also examined in all animals of the low and mid dose groups)
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 was considered as statistically significant).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
dose-dependent clinical signs associated with local irritation of the test item
Mortality:
mortality observed, treatment-related
Description (incidence):
dose-dependent clinical signs associated with local irritation of the test item
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significantly higher kidney weights (absolute weight 16%, relative weight 15% above controls) in high dose group males (600 mg/kg bw/day)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day: multifocal tubular degeneration/regeneration, tubular dilation + crystalline deposits in altered tubules, focally extensive transitional cell hyperplasia in renal pelvis, focally extensive transitional cell hyperplasia of urinary bladder
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One low dose group female (75 mg/kg bw/day) was found dead on Study day 24. In view of its isolated occurrence and lack of dose relationship this death was not considered to be test item-related.
Male and female animals of the high dose group (600 mg/kg bw/day) moved the bedding immediately after administration. This was mostly associated with slight or moderate piloerection and later also slight, moderate or – in few animals severe salivation at the time of administration. These observations can be ascribed to a local irritant effect of the test item. Moving the bedding and slight piloerection were also noted after administration occasionally in mid dose group animals (250 mg/kg bw/day), and slight signs of irritation (moving the bedding and salivation) were also observed in a single female animal of the low dose group (75 mg/kg bw/day) on a single day. Signs of local irritation were not observed during the recovery period. Slight piloerection was only seen in few animals on the first days of the recovery period.
Observations related to a lesion during gavaging (emesis, severe piloerection and half eye-lid closure, slightly increased spontaneous activity and abnormal breathing, vocalization, severe salivation and hypothermia) were noted in 3 animals of the high dose group and were thus concluded to be not test substance-related.
Occasional isolated findings, like red nasal discharge or eschar were also observed in control animals and are not assumed to be related to the test item.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

BODY WEIGHT AND WEIGHT GAIN
Throughout the treatment and recovery period, body weights were within the normal range of variation for this strain. Body weight gain was not considerably different between the groups and neither for male nor for female animals were there any significant differences in body weight between the dose groups and the control group of this study.

FOOD CONSUMPTION
No significant difference in food consumption was found in the treatment or recovery period between the dose groups and the control group of both genders.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings in any of the animals of this study.

HAEMATOLOGY
The test item had no effect on haematological parameters and coagulation parameters analysed at the end of the treatment and recovery period. There were no toxicologically relevant differences in any of the parameters between dose groups and control group. Slightly – but statistically significantly higher platelet count and prolonged activated partial thromboplastin time (aPTT) in male animals of the high dose group (600 mg/kg bw/day) at the end of the recovery period and of large unstained cells of female animals of the high dose group (600 mg/kg bw/day) at the end of the treatment period were in the normal range of historical data and are not assumed to be biologically relevant.

CLINICAL CHEMISTRY
The test item had no effect on clinical biochemistry markers analysed at the end of the treatment and recovery period. There were no toxicologically relevant differences in any of the parameters between dose groups and control group. Slightly – but statistically significantly lower albumin levels in male animals of all dose groups observed at the end of the treatment period – but not in high dose group animals (600 mg/kg bw/day) at the end of the recovery period are not assumed to be toxicologically relevant. Statistically significantly lower total bilirubin levels in female – but not male animals of the high dose group (600 mg/kg bw/day) at the end of the recovery period are not assumed to be toxicologically relevant.

URINALYSIS
The test item had no effect on urinary parameters analysed at the end of the treatment and recovery period. A high count of red blood cells was found in the urine of a single female each of the mid dose group (250 mg/kg bw/day) at the end of the treatment period and of the high dose group (600 mg/kg bw/day) at the end of the recovery period.

NEUROBEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

ORGAN WEIGHTS
At the end of the treatment period a slight and statistically significantly higher kidney weight (absolute weight 16%, relative weight 15% above controls) was found in male animals of the high dose group (600 mg/kg bw/day) and was associated with histopathological findings in the kidney of these animals.
A slightly – but not statistically significantly lower thymus weight (21% below controls) in female – but not male animals of the high dose group (600 mg(kg bw/day) is not considered to be the result of a primary toxicological effect of the test item. When compared to controls, absolute thyroid weight of female animals of the mid dose group (250 mg/kg bw/day; 22% below controls) and high dose group (600 mg/kg bw/day; 27% below controls and statistically significant) was slightly lower than in controls. As this is in contrast to thyroid weight of male animals, where slightly higher thyroid weight was found in mid dose group (250 mg/kg bw/day; 27% above controls) and high dose group (600 mg/kg bw/day; 19% above controls), this is not assumed to be a test item related effect. Slight but statistically significant differences in absolute brain weight of male animals of the low dose (75 mg/kg bw/day) and high dose (600 mg/kg bw/day) groups are not assumed to be toxicologically relevant as values were within the normal range of historical data. At the end of the recovery period, relative kidney weight (to brain weight) of female animals of the high dose group (600 mg/kg bw/day) was slightly (16% above controls) and statistically significantly higher than in controls. Besides, there were no considerably altered organ weights in male and female animals. Statistically significantly higher heart weight of animals of the high dose group (600 mg/kg bw/day) and pituitary gland weight in male high dose group animals (600 mg/kg bw/day) is not considered toxicologically relevant as the values were within the normal range of historical data.

GROSS PATHOLOGY
Only single specific gross pathological changes were recorded for male and female animals at the end of the treatment period and are not considered to be treatment-related. These single findings were a yellow spot on one epididymis and a fluid-filled uterus in a control and high dose (600 mg/kg bw/day) animal, respectively, discoloured red axillary lymph nodes and thymus in one female animal of the low dose group (75 mg/kg bw/day), dark discoloured lungs in another low dose animal (75 mg/kg bw/day), one whitish focus on one adrenal gland and kidney of an mid dose animal (250 mg/kg bw/day) and an uterus distended with fluid. A fluid-filled uterus was also found in one control animal at the end of the recovery period. According to the authors, the above-mentioned alterations were incidental findings and were not assumed to be related to the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
No histopathological findings were noted that could be associated with the death of the low dose female (75 mg/kg bw/day) found dead. In view of its isolated occurrence and lack of dose relationship this death was not considered to be test item-related.
Test item-related histopathological findings considered to be toxicologically relevant were seen in the kidney and urinary bladder. In the kidney, minimal to moderate multifocal tubular degeneration/regeneration was observed in all high dose group males and females (600 mg/kg bw/day), in all males accompanied by minimal tubular dilation and in most animals showing crystalline deposits within altered tubules. In one single female of this dose group, a minimal focally extensive transitional cell hyperplasia was seen in the renal pelvis. In the urinary bladder focally extensive transitional cell hyperplasia was also noted in two animals of the high dose group (600 mg/kg bw/day). Renal and urinary bladder changes noted in high dose animals (600 mg/kg bw/day) were not reversible after the 14-day recovery period and were considered to be adverse. Minimal multifocal tubular degeneration/ regeneration, noted in 2/5 mid dose group males (250 mg/kg bw/day) at terminal sacrifice, was not considered adverse as it was not accompanied by any changes in clinical pathology or organ weight parameters.
Furthermore, at terminal sacrifice there was indication of a local irritant effect of the test item formulation to the gastric epithelium, predominantly comprising diffuse hyperplasia of the nonglandular epithelium and/or epithelial hyperplasia of the limiting ridge in both sexes of the high dose group (600 mg/kg bw/day) and in females of the mid dose group (250 mg/kg bw/day). These lesions did not resolve during the recovery period. In the glandular part of the stomach, an increased incidence and severity of submucosal mixed cell infiltrate(s) and some minor (multi)focal mucosal congestion/haemorrhage were noted in the high dose group (600 mg/kg bw/day), but were no longer seen after the recovery period. As gastric lesions were related to the specific administration route used in this study, they were considered to be irrelevant for risk assessment in humans. Likewise, in the lung, multifocal minimal or mild chronic (peri)bronchitis was noted in two high dose males (600 mg/kg bw/day), at terminal sacrifice, and was considered to indicate a local irritant effect of the test item formulation, when accidentally partially instilled into the lung.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April 2019 - 08 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 25 June 2018)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted August 1998)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008, L 142, Annex Part B
Version / remarks:
(30 May 2008)
Qualifier:
according to
Guideline:
other: Commission Directive 2001/59/EC
Version / remarks:
(6 August 2001)
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no
Specific details on test material used for the study:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Before the beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about concentration (±10 %) and stability of the test item in the selected vehicle.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
As recommended by current guideline.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7 - 8 weeks old
- Weight at study initiation: males: 295 – 353 g; females: 187 – 230 g
- Fasting period before study: no
- Housing: full barrier in an air-conditioned room; in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany; ad libitum)
- Water: tap water in bottles ad libitum, sulphur acidified to a pH of approximately 2.8
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: The acidification of the tap water is to provide additional protection against microbial contamination; municipal residue control and microbiological controls of the drinking water at regular intervals; certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
other: sterile water (aqua ad injectionem)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared at least every 10 days. Dose volumes were adjusted individually based on weekly body weight measurements. The test item was ground into a fine powder and weighed into a tared plastic vial on a precision balance. Dose formulations were prepared by adding the required volume of vehicle (aqua ad injectionem) to obtain the appropriate final concentration (w/v) of the test item. Prepared formulations were vortexed and / or stirred until visual solubility was achieved. The prepared formulation was stored protected from light, at room temperature. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 10, 30 and 50 mg/mL (low, mid and high dose group, respectively)
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: 901110 and 902272
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Investigation of homogeneity and substance concentration was included on samples from various levels (top, middle and bottom) of prepared formulations from all dose groups and from the middle of the control group in study week 1, 5, 9 and in the last week of treatment (40 samples). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 1 mL). All A-samples and B-samples of week 5 were analysed at CIP Chemisches Institut Pforzheim GmbH (Eurofins Munich, Study Phase No. 186793) and until then stored under appropriate conditions based on available stability data. Remaining B-samples were retained at < -15 °C at CIP Chemisches Institut Pforzheim GmbH and discarded after completion of the final study report.
Dose formulations were considered homogeneous due to relative standard deviations of the respective formulation concentration, which were below 10% (except samples 2a, 3a and 4a (week 1 low dose), which showed a RSD of 16.9%).
Recoveries of test item in test item formulations were found between 62.0 and 154.0%. Test item concentrations ± 10% in comparison to the nominal concentrations are regarded to be acceptable. This requirement is fulfilled for the samples 3a, 18a, 19a, 22a to 27a, 29a and 30a. In week one (samples 1a to 10a), test item concentrations higher than targeted were determined. Subsequent investigations revealed that a recently calibrated pipette was still malfunctioning, leading to a sample volume greater than intended. These values were excluded from analyses. Samples of week 5 and 13 were below the acceptable range. Subsequent investigations revealed that the test item degrades in contact with oxygen explaining lower test item recoveries. Hence, actually achieved concentrations were recalculated based on the lowest mean values achieved for each dose level at the different testing occasions, resulting in concentrations of 32, 103 and 311 mg/kg bw/day.
The ongoing validity of the analytical method was verified using procedural recoveries prepared with test item “CIP Code 11225”, that had previously been stored at BSL for several months. When using this test item, mean recovery was 49 % and 56 % respectively. Recoveries from 07 June to 12 August 2019 did not fulfil the SANCO 3029/99 rev. 4 requirements that recoveries should be in the range 70 - 110% (ideally 80% - 100%).
Therefore, to verify the analytical method, a second test was performed under non-GLP using test item received for pre-tests from Eurofins Munich / BSL Munich. The recoveries were found at 94% and 100% and therefore, fulfilled the requirements of SANCO 3029/99 rev. 4.
The analytical method was additionally confirmed by analysing recovery samples using certified ammonium niobium oxalate reference item (CIP Code 11198). The recoveries were found at 98 % and 103 % and therefore, fulfilled the requirements of SANCO 3029/99 rev. 4.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
32 mg/kg bw/day (actual dose received)
Remarks:
correspond to a nominal dose of 50 mg/kg bw/day
Dose / conc.:
103 mg/kg bw/day (actual dose received)
Remarks:
correspond to a nominal dose of 150 mg/kg bw/day
Dose / conc.:
311 mg/kg bw/day (actual dose received)
Remarks:
correspond to a nominal dose of 450 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose groups were selected according to the results of a previous dose range finding study (BSL Munich, Study No. 123978, non-GLP) and a 28-day repeated dose oral toxicity study (BSL Munich Study No. 123979, GLP) as well as in consultation with the sponsor. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response. In the 14-day dose range finding oral toxicity study with Ammonium Niobium Oxalate in Wistar rats mortality of 2/3 females at 1000 mg/kg bw/day was observed. Thus, 1000 mg/kg bw/day was not considered as a feasible dose for the present study and 450 mg/kg bw/day was defined as the highest dose level for the oral 90-day repeated dose toxicity study in Wistar rats.
- Fasting period before blood sampling for clinical biochemistry: overnight
- Rationale for selecting satellite groups: no satellite groups
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Inadvertently, clinical signs were not recorded on the 10th and 28th of May. Inadvertently, detailed cage side observations were not recorded in week 10 for mid dose male no. 27 and high dose males no. 36 and 38.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first administration and at least once a week thereafter.
Detailed clinical observations considered, but were not limited to, spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period. In addition, body weight gain was also assessed weekly during the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was measured weekly during the treatment period and calculated as g food/animal/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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the first administration and in the last week of the treatment period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period.
- Anaesthetic used for blood collection: Yes (ketamine / xylazine; terminal anaesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), glucose (Gluc), sodium (Na), potassium (K). For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (78 animals) and stored at < -15 °C. T3, T4, TSH, LDL and HDL serum levels were determined of all animals.

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to or as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: urine colour / appearance, specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leuc)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and once in the last week of exposure.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
EXAMINATION OF FERTILITY PARAMETERS:
During the last two weeks of treatment, the estrous cycle of all female animals was monitored.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed, necropsied and examined post mortem. The detailed gross pathology included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.
The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Thyroid / parathyroid glands were weighed after fixation. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded, except for animal no. 78, which was already sacrificed on study day 90 (last day of test substance administration). Data of this animal were not included in statistical analyses, but were listed in the result tables.
Organs weighed: liver, uterus with cervix, kidneys, thymus, adrenals, thyroid / parathyroid glands (weighed after fixation), testes, spleen, epididymides, brain, prostate with seminal vesicles and
coagulating glands, pituitary gland, ovaries, heart

Left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters.

HISTOPATHOLOGY: Yes
A full histopathological evaluation of tissues was performed on high dose and control animals and any animal found dead or euthanised before the planned day of sacrifice. For testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides. For organs and tissues showing treatment-related changes (kidneys and urinary bladder) in the high dose group, these examinations were extended to animals of all other dosage groups. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

Tissues examined: adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular, parotis), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland including parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina.
Other examinations:
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0). Sperm morphology slides were prepared from all male animals.
Statistics:
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage. All results are reported in a tabular form (summarised in mean or summary tables and / or listed in individual data tables).
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett’s Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation in 1/20 low dose animals, 4/20 mid dose animals and in 15/20 high dose animals as well as moving the bedding in 13/20 high dose animals are deemed to be a local reaction to the test item. No such effects was seen in the control group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
3 (2 males and 1 female) artefactual mortalities occurred in the high dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose males exhibited statistical significantly lower body weights compared to controls on study day 43 (-8.28%) and 50 (-7.63%). These findings are considered as test item-related, and toxicologically relevant. In accordance with these findings, high dose males exhibited a statistically significantly reduced body weight gain (91.25 g) compared to controls (120.10 g) over the whole study period. No such effects were seen in females.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Platelet counts were statistically significantly increased in high dose animals compared to control animals (males: 823.00 x 10°9 cells/L vs. 696.40 x 10^9 cells/L in controls and females: 817.56 x 10^9 cells/L vs. 696.30 x 10^9 cells/L in controls). It cannot fully be excluded that this effect is test item related, it is however not considered toxicologically relevant due to the small size of the change and because all other haematological parameters were inconspicuous, i.e., without effects.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In mid dose and high dose males as well as in high dose females the urinary pH was slightly lower than in controls. Moreover, 1/8 mid dose, 2/7 high dose males and 4/10 high dose females exhibited elevated urinary glucose levels (20 mg/dL) compared to the animals of the control and low dose group. Erythrocytes were increased in 4/10 high dose males. The amount of protein was also marginally increased in high dose animals compared to corresponding controls.
Moreover, 1/8 mid dose, 2/7 high dose males and 4/10 high dose females exhibited presence of urinary glucose (20 mg/dL) compared to the animals of the control and low dose group, which were all negative. The amount of erythrocytes/blood cells in the urine was also increased in 4/10 high dose males. In two of these males, little urine was available and it was hazy and brownish (male no. 35) or reddish (male no. 31). The amount of protein was also marginally increased in high dose animals compared to corresponding controls. No further abnormalities were detected in the other urinary parameters.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Spontaneous activity (mean score: 3.70 vs. mean score 4.00 of controls) and urination were statistically significantly reduced in high dose animals compared to corresponding controls (mean score: 3.70) compared to control males (mean score: 4.00).and it cannot fully be excluded that these reductions are correlated to the test item.
Spontaneous activity of high dose males and females was statistically significantly reduced compared to that of corresponding controls (mean score males: 3.00 vs. control: 4.00, mean score females: 3.10 vs. 4.00 in controls). However, dose-dependency was only observed in females. Although, high dose males already exhibited reduced spontaneous activity during pre-treatment measurements, effects seen in the last study week are considered as test item related as differences were stronger and apparent in both sexes.
Urination was dose-dependently reduced in test item treated males and females compared to corresponding controls. The difference between high dose and control females reached statistical significance. In accordance with macroscopic and histopathological findings, this effect is considered as test item related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean kidney weight, kidney to body weight and kidney to brain weight ratios were increased in high dose animals (males and females) compared to controls. Similar increases could be seen in high dose females. The difference in kidney to brain weight ratio reached statistical significance (high dose: 0.7445% vs. 0.6847% in controls).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
An abnormal granular surface and / or a pale colour of the kidneys was recorded in 7/10 high dose males and 4/10 high dose females.
Neuropathological findings:
no effects observed
Description (incidence and severity):
3 Male no. 33 (high dose group) had to be euthanised on study day 64 after misadministration with the following clinical signs: reduced spontaneous activity, piloerection, half eyelid closure and abnormal breathing. Male no. 34 (high dose group) had to be euthanised on study day 14 with paresis of the right hindlimb because of a dislocated hip. Female no. 78 (high dose group) exhibited abnormal breathing and piloerection shortly after administration on study day 89. Most probably some test material attained the nasal passages and lungs due to reflux. The animal therefore underwent normal necropsy one day before its originally scheduled necropsy (study 90 instead of study day 91).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, renal changes consisting of intratubular deposition of pale yellow, translucent, angular, anisotropic crystals (due to the nature of the test item most probably calcium oxalate crystals) with subsequent nephropathy. No such findings were reported for the cotrol, the low dose and the mid dose groups.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, treatment with Ammonium Niobium Oxalate led to transitional cell hyperplasia of the urinary bladder epithelium. No such findings were reported for the cotrol, the low dose and the mid dose groups.
Other effects:
no effects observed
Details on results:
- Mortality:
All animals of the control group as well as the low and mid dose treated groups survived until scheduled time of sacrifice. In the high dose group, no treatment-related mortalities were noticed, however, 3 cases (2 males and one female) of incidental death were reported.
Male no. 33 (high dose group) had to be euthanised on study day 64 after misadministration. Male no. 34 (high dose group) had to be euthanised on study day 14 with paresis of the right hindlimb because of a dislocated hip. Female no. 78 (high dose group) exhibited abnormal breathing and piloerection shortly after administration on study day 89. Most probably some test material attained the nasal passages and lungs due to reflux. The animal therefore underwent normal necropsy one day before its originally scheduled necropsy (study 90 instead of study day 91).

- Clinical observations (Please also refer to Tables 1 - 4):
CONTROL:
No treatment related clinical signs were recorded. A scratch followed by a crust on the lower back of female no. 50 from study day 64 until the end of the study was considered as incidental.
LOW DOSE:
A crust on the neck of male no. 13 on study days 47 and 48 was considered as incidental. Increased aggressive behaviour in male no. 19 from study day 14 to 20 and in female no. 55 from study day 13 to 19 is rather a consequence of discomfort induced by treatment with the test item, but is not considered as toxicologically relevant. Slightly increased salivation was observed in male no. 15 on study day 42 and is deemed to be a local reaction to the test item. Statistically significant differences in parameters of detailed clinical observations of week 4 (animal sleeps and animal moving around in cage) are considered as incidental.
MID DOSE:
Increased aggressiveness and slightly increased salivation were recorded for male no. 23 from study day 15 to 21 and 46 to 67 and for male no. 27 from study day 14 to 20 and 45 to 56. Severe salivation was observed in male no. 25 between study days 39 to 41. Slight erythema was observed in male no. 26 from study days 45 until 56. In females, hairless areas on both forelimbs were seen in no. 61 on study day 1 and subsequently between study days 11 and 19. Moreover, female no. 70 exhibited moderately to severely increased salivation from study day 29 until the end of the study.
HIGH DOSE:
Moving the bedding was observed in 10/10 males and 3/10 females, while 8/10 males and 7/10 females exhibited increased salivation.
Detailed clinical observations of week 8, 10, 11 and 12 were in accordance as salivation was statistically significantly increased in high dose males compared to controls (week 8: +22.2%, +21.4%, week 11: +43.8%, week 12: +18.8%). For females, detailed clinical observations of week 5, 10 and 12 also displayed increased salivation in high dose animals compared to controls (week 5: +25%, week 10: +30%, week 12: +25%).
Statistically significant differences regarding the parameters “animal sleeps” and “animal moving around in cage” of detailed clinical observations of females of all dose groups in week 12 as well as the statistically significantly increased response to handling in high dose females in week 5 are considered as incidental.
Besides the clinical findings mentioned above, moderately reduced spontaneous activity (study day 18 to 20) and a scratch on the snout (study day 87 to 90) were recorded in high dose male no. 36. In high dose male no. 38, abnormal breathing (study day 26) and moderately reduced spontaneous activity (study day 28 until 30) were observed. Due to reflux, some test material might have attained the nasal passages and lungs, resulting in the just mentioned clinical symptoms. High dose female no. 78 had in addition hairless areas on the right forelimb on study day 43 to 66, on the abdomen from study day 55 until 66 as well as on the neck and upper back on study day 67 until the end of the study.
As formerly mentioned, increased aggressiveness and moving the bedding are rather a consequence of the adverse experience (e.g. due to bad taste) of being dosed with the test item, but are not considered as toxicologically relevant. Findings regarding the skin and fur are considered as incidental, while increased salivation showed dose-dependency, but is deemed to be a local reaction to the test item.

- Functional Observation Battery and Ophthalmology (Please also refer to Tables 5 - 6):
Pre-Values:
Within the low dose group statistically significantly fewer males slept during the observation period compared to control males (mean score: 0.50 vs. control: 1.00). In accordance, more low dose males moved around in their cages during the observation period compared to control males (mean score: 0.50 vs. control: 0.00). Moreover, high dose males exhibited statistically significantly reduced spontaneous activity (mean score: 3.70) compared to control males (mean score: 4.00).
Statistically significantly more high dose females slept during the observation time compared to control females (mean score: 0.50 vs. control: 0.00). Accordingly, fewer high dose females were moving in their cages compared to control females during the observation period (mean score: 0.50 vs. control: 1.00).
All of these findings are within normal background variation and were observed before dosing.

Last Study Week:
Spontaneous activity of high dose males and females was statistically significantly reduced compared to that of corresponding controls (mean score males: 3.00 vs. control: 4.00, mean score females: 3.10 vs. 4.00 in controls). However, dose-dependency was only observed in females. Although, high dose males already exhibited reduced spontaneous activity during pre-treatment measurements, effects seen in the last study week are considered as test item related as differences were stronger and apparent in both sexes.
Urination was dose-dependently reduced in test item treated males and females compared to corresponding controls:
Mean scores male: high dose: 0.38, mid dose: 0.40, low dose: 0.50, control: 0.80
Mean scores female: high dose: 0.30, mid dose: 0.50, low dose: 1.00, control: 1.10
The difference between high dose and control females reached statistical significance. In accordance with macroscopic and histopathological findings, this effect is considered as test item related.
Test item treated males exhibited a less defensive behaviour towards handling than control males. This effect was dose-dependent, yet statistically not significant (mean scores high dose: 3.00, mid dose: 3.70, low dose: 3.80, control: 3.80). In accordance with this, the arousal and fear response of test item treated males was dose-dependently decreased, yet again not statistically significantly (mean scores arousal high dose: 3.75, mid dose: 3.80, low dose: 4.00, control: 4.00; mean scores fear high dose: 3.75, mid dose: 4.00, low dose: 4.20, control: 4.20).
Similar to males, test item treated females exhibited less defensive behaviour towards handling compared to control females. This effect was dose-dependent and in high dose females this difference reached statistical significance (mean scores high dose: 2.60, mid dose: 3.00, low dose: 4.00, control: 4.40). However, no differences in fear or arousal could be observed.
Defecation was dose-dependently increased in test item treated males (mean scores high dose: 1.38, mid dose: 1.30, low dose: 1.10, control: 0.80). This finding is not considered as toxicologically relevant

- Body Weight Development (Please also refer to Tables 7 - 9):
Overall the mean body weight increased normally during the study in control as well as in all dose groups.
Slightly reduced body weights on the day of sacrifice (study day 91) are an effect of overnight fasting.
Mid dose males exhibited marginally, non-significantly lower body weights compared to control males from study day 15 until the end of the study. Deviations versus control ranged from -0.51% up to -3.59%. This effect is considered as test item-related. However, changes were so small that they were not considered as toxicologically relevant. A similar effect was observed in high dose males, but reductions were stronger than those seen in mid dose males (deviation vs. control ranging from -5.69% up to -8.28%). On study day 43 and 50 differences in body weights of high dose males (day 43: 394.2 g and day 50: 404.1 g) compared to control males (day 43: 429.8 g and day 50: 437.5 g) reached statistical significance. No such effects were seen in females. This effect observed in males is considered as test item related and toxicologically relevant.
In accordance with the above mentioned findings, high dose males exhibited a statistically significantly reduced body weight gain (91.25 g) compared to controls (120.10 g) over the whole study period. Remaining statistically significant differences in body weight gain in week 1, 2 and 9 are considered as incidental and of no toxicological relevance.

- Food Consumption:
Overall no treatment-related effects on food consumption were observed during the treatment period.

- Fertility Parameters:
Overall no treatment-related effects on fertility parameters (estrous cycle, sperm count, sperm motility and morphology) were observed during the treatment period of the study in control as well as in all dose groups. Occasional statistical significant differences concerning sperm morphology are not considered as toxicologically relevant as the amount of abnormal sperms was lower in high dose males than in controls.

- Haematology and Blood Coagulation (Please also refer to Tables 10 - 11):
Statistically significantly increased platelet (PLT) counts were observed in high dose animals compared to controls (males: 823.00 x 10^9 cells/L vs. 696.40 x 10^9 cells/L in controls and females: 817.56 x 10^9 cells/L vs. 696.30 x 10^9 cells/L in controls). Although it cannot fully be excluded that this effect is test item related, it is not considered as toxicologically relevant as differences were marginal.
Eosinophil (Eos) percentages were slightly increased in test item treated males compared to controls. Control: 0.4100%, low dose: 0.8111%, mid dose: 0.5857%, high dose: 0.6167%; historical control data: 0.5%, SD 0.4, range: 0.0 - 3.2. In females no increase could be observed. Noticeably high eosinophil percentages in low dose females can be explained by one very high value in female no. 60 (7.10%). As no dose-dependence could be detected and differences were only marginal, these effects are considered as not toxicologically relevant.
Slightly decreased neutrophil percentages could be seen in mid dose and high dose animals compared to corresponding controls (males mid dose: 24.229% and high dose: 22.183% vs. 27.340% in controls; historical control data: 21.3%, SD 7.6, range: 6.4 - 84.0; females mid dose: 19.850% and high dose: 17.544% vs. 24.170% in controls; historical control data: 18.7%, SD 10.9, range: 5.0 - 88.9). Marginally increased monocyte percentages were recorded in test item treated males (low dose: 3.033%, mid dose: 2.757%, high dose: 2.500%) compared to controls (2.000%; historical control data: 2.5%, SD 1.4, range: 0.9 - 19.0). In the low dose and mid dose groups differences reached statistical significance. In females, a similar dose-dependent, yet non-significant increase could be observed. Control: 2.21%, low dose: 2.32%, mid dose: 2.47%, high dose: 2.91%; historical control data: 2.2%, SD 1.2, range: 0.3 - 13.5.
Effects in eosinophils, neutrophils and monocytes were all marginal and still within the normal variation range. Therefore they are considered as not toxicologically relevant.
No treatment-related effects on coagulation were observed in any of the dose groups.

- Clinical Biochemistry and Thyroid Hormone Determination:
No treatment-related effects on thyroid hormones (T3, T4 and TSH) or parameters of clinical biochemistry were determined in any of the dose groups.
Statistically significantly reduced ALAT and ASAT levels were observed in high dose males (ALAT: 28.638 U/L, ASAT: 81.462 U/L) compared to controls (ALAT: 47.510 U/L, deviation vs. control -39.723%, ASAT: 125.950 U/L, deviation vs. control -35.322%). In females no effects on ALAT were observed, but ASAT levels were also reduced in mid dose and high dose females compared to controls (control: 94.75 U/L, MD: 67.47 U/L, HD: 72.78 U/L). In the mid dose group reduction was statistically significant (-28.8%).
Historical control data ALAT (males): 38.1 U/L, SD 67.2, range: 8.1 - 1365.0
Historical control data ALAT (females): 30.5 U/L, SD 11.6, range: 4.8 - 129.8
Historical control data ASAT (males): 97.0 U/L, SD 82.4, range: 16.4 - 1667.0
Historical control data ASAT (females): 86.8 U/L, SD 24.8, range: 27.9 - 195.1
In addition, marginally, but statistically significantly reduced levels of total protein (TP), albumin (Alb) and total cholesterol (Chol) were determined in the male high dose group compared to corresponding controls:
TP: control: 59.420 g/L, high dose: 52.925 g/L; historical control data: 55.6 g/L, SD 5.8, range: 27.5 - 75.6
Alb: control: 33.033 g/L, high dose: 30.138 g/L; historical control data: 31.0 g/L, SD 3.6, range: 15.3 - 41.0
Chol: control 1.626 mmol/L, high dose: 1.950 mmol/L; historical control data: 1.7 mmol/L, SD 0.4, range: 0.8 - 3.8
Similar statistically significant reductions in albumin and total protein could be determined in high dose females compared to control females:
TP: control: 60.38 g/L, high dose: 54.622 g/L; historical control data: 59.3 g/L, SD 6.6, range: 3.8 - 75.1
Alb: control: 36.496 g/L, high dose: 33.8178 g/L; historical control data: 34.6 g/L, SD 4.3, range: 16.9 - 44.9
Creatinine levels were statistically significantly reduced in mid dose and high dose females compared to controls (control: 29.60 µmol/L, mid dose: 25.80 µmol/L, high dose: 25.56 µmol/L; historical control data: 28.5 µmol/L, SD 9.9, 7.0 - 131.0). In males this effect was not observed.
Sodium (Na) concentrations were slightly, but statistically significantly reduced in mid dose as well as in high dose males compared to controls (mid dose: 140.70 mmol/L, high dose: 138.38 mmol/L vs. 142.60 mmol/L in controls; historical control data: 135.6 mmol/L, SD 15.8, range: 56.0 - 169.0). No such effects could be observed in females.
Triglyceride (TG) concentrations were higher in test item treated males and females compared to corresponding controls:
Control male: 0.517 mmol/L, low dose: 0.684 mmol/L, mid dose: 0.762 mmol/L, high dose: 0.778 mmol/L; historical control data: 0.8 mmol/L, SD 0.4, range: 0.2 - 2.1
Control female: 0.337 mmol/L, low dose: 0.405 mmol/L, mid dose: 0.573 mmol/L, high dose: 0.516 mmol/L; historical control data: 0.5 mmol/L, SD 0.3, range: 0.1 - 1.6
Differences were not statistically significant and mean values were still within normal background variation. Therefore, differences are considered as not toxicologically relevant.
Reductions in the parameters mentioned above are not considered as toxicologically relevant.
Of note, values of alkaline phosphatase, sodium and potassium of high dose female no. 75 are outliers according to the outlier tests of Grubbs, Dixon and Nalimov.

- Urinalysis:
In mid dose and high dose males the urinary pH was slightly lower (mean of 6.2) than in control or low dose males (mean of 7.1). A similar effect was observed in high dose females compared to the other dose groups (mean pH high dose: 6.2 vs. mean pH control: 7.1). Moreover, 1/8 mid dose, 2/7 high dose males and 4/10 high dose females exhibited presence of urinary glucose (20 mg/dL) compared to the animals of the control and low dose, which were all negative. The amount of erythrocytes/blood cells in the urine was also increased in the urine of 4/10 high dose males. In two of these males, little urine was available and it was hazy and brownish (male no. 35) or reddish (male no. 31). The amount of protein was also marginally increased in high dose animals compared to corresponding controls (2 animals with 30 mg/dL, 4 animals with 100 mg/dL and 1 animal with 500 mg/dL compared to 4 animals with 30 mg/dL and 2 animals with 100 mg/dL for the controls). No further abnormalities were detected in the other urinary parameters.

- Pathology (Please also refer to Tables 12 - 13):
CONTROL:
In control male no. 1 and female no. 42 the right axillary lymph nodes and in female no. 43 the mandibular lymph nodes had an abnormal red colour, which are considered as incidental findings. The lungs of male no. 4 exhibited several beige and the thymus of male no. 2 several red foci, the latter due to hemorrhages. In male no. 10 a smaller, atrophied right seminal vesicle was recorded. In female no. 41 the thymus had an abnormal red colour and in female no. 42 a red focus of 0.4 cm was detected in the thymus, both due to hemorrhages. In females no. 41, 45, 47 and 48 both uterus horns were dilated, which is related to the estrous cycle and not considered as treatment related.

LOW DOSE:
An abnormal red colour due to hemorrhage was observed for the mediastinal lymph nodes of male no. 11, the thymi of males no. 14 and 15, the jejunum and ileum of male no. 20 as well as the ileum of female no. 56. In addition, the urinary bladder of female no. 56 also showed an abnormal red colour due to congestion. In female no. 51 several red foci were detected in the thymus. Females no. 51 and 52 exhibited a dilatation of the uterus, which is related to the estrous cycle and not considered as test item related.

MID DOSE:
An abnormal red colour was recorded for the right seminal vesicles of male no. 24, the ileum of male no. 25, the thymus of male no. 28 as well as the left axillary lymph nodes of male no. 30, the jejunum of female no. 68 and the medulla of the thymus of female no. 64. In male no. 29 a dilatation of the right kidney was observed. In male no. 27 the left testis and the left epididymidis were absent. Male no. 26 exhibited small, atrophied prostate glands and small seminal vesicles. In female no. 61 a dilatation of the uterus was observed, which is again not considered as test item related, but related to the estrous cycle.

HIGH DOSE:
An abnormal granular surface and / or a pale colour of the kidneys was recorded for males no. 31, 32, 33, 35, 36, 38 and 40 as well as for females no. 71, 74, 75, 79. In female no. 79 additionally a focus was detected on the left kidney. These findings are in accordance with histopathological results and considered as test item related.
In absence of histopathological correlation, the following macroscopic findings during necropsy are considered as incidental and not test item related or toxicologically relevant:
An abnormal red colour of the ileum was observed in males no. 39 and 40 as well as in females no. 71 and 74. In female no. 71 mesenteric lymph nodes also exhibited abnormal red colour.
In prematurely euthanized male no. 33 the entire stomach was thick and white, the mucosa of the duodenum, jejunum and ileum was thick and in the thymus several red foci of 0.1-0.2 cm were detected.
In male no. 36 the right hemisphere of the brain was fluid filled and the pituitary gland was absent. Moreover, a red focal area within the stomach was observed. The thymus exhibited multiple red foci and mandibular lymph nodes had an abnormal red colour.
In male no. 32 one red fundic area, while in male no. 39, few (2-6) red fundic areas were recorded within the stomach. Moreover, few red foci of 0.1 cm were found in the thymus of male no. 39 and one red focus of 3mm was found in the thymus of male no. 37.
Dilatation of the uteri was observed in females no. 74 and 75. These findings are not test item related, but related to the estrous cycle.

- Organ Weights (Please also refer to Tables 14 - 15):
Mean kidney weight, kidney to body weight and kidney to brain weight ratios were statistically significantly increased in high dose males (organ weight: 3.0614 g, organ / body: 0.7102%, organ / brain: 145.5157%) compared to controls (organ weight: 2.5835 g, organ / body: 0.5714%, organ / brain: 117.6647%). Similar increases could be seen in high dose females. The difference in kidney to brain weight ratio reached statistical significance (high dose: 0.7445% vs. 0.6847% in controls).
The above mentioned effects are in accordance with histopathological findings in this organ and are considered as test item related.
A statistically significantly reduced heart to body weight ratio was determined in low dose males (0.2581%) compared to controls (0.2787%). In contrast, high dose females exhibited statistically significantly increased heart to body weight ratio in comparison to controls (0.3546% vs. 0.3289% in controls). In both cases, differences were marginal and values were still within normal background variation and are therefore not considered as test item related.
Mean ovaries to body weight ratio as well as ovaries to brain weight ratio was statistically significantly increased in hig dose females (organ / body: 0.0568%, organ / brain: 6.8352%) compared to controls (organ / body: 0.0483%, organ / brain: 5.8292%). Again, differences were marginal and values were still within normal background variation. This finding is hence not considered as test item related.

- Histopathology
Test item related changes were observed in kidneys of males and females from the high dose group. These renal changes consisted of intratubular deposition of pale yellow, translucent, angular, anisotropic crystals (due to the nature of the test item most probably calcium oxalate crystals) and subsequent nephropathy characterized by tubular necrosis, tubular basophilia, interstitial fibrosis, mononuclear infiltrates, hyaline casts, tubular dilatation and hyperplasia of the transitional epithelium of the renal pelvis. Further, transitional cell hyperplasia was observed in the urinary bladder from several high dose group animals. Observed changes in the kidneys and the urinary bladder were of higher incidence and severity in males when compared to females.
Under the condition of this study treatment with Ammonium Niobium Oxalate caused renal changes (oxalate nephropathy) and transitional cell hyperplasia of the urinary bladder epithelium in males and females at 311 mg/kg bw/day only.
Key result
Dose descriptor:
NOAEL
Effect level:
103 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
311 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
103 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (reliability 1 and 2). The information is suitable for hazard assessment leading to an endpoint conclusion and is therefore sufficient to fulfil the standard information requirements set out in Annex IX, 8.6., in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
System:
urinary
Organ:
bladder
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Reliable data are available for the oral route.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The registered substance is classified as irritating to respiratory tract (STOT Single Exp. 3, H335) based on the results from an acute inhalation toxicity study and its physico-chemical properties (refer to acute toxicity section for further details). Therefore, local effects to the respiratory tract are also expected after prolonged or repeated exposure.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Reliable data are available for the oral route.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Reliable data are available for the oral route.

Additional information

Repeated Dose Toxicity: via oral route

A reliable key repeated dose rat toxicity study with Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate is available for the oral route, conducted according to OECD test guideline 407 (subacute exposure duration), and in compliance with GLP (CBMM Europe BV, Key, 2014, RDT). Five Wistar rats per sex per dose received daily oral gavages of the test item dissolved in water at doses of 75, 250 and 600 mg/kg bw/day for 28 consecutive days. The doses selected were based on the results of a previously conducted 14-day range-finding study (CBMM Europe BV, Supporting, 2013, DRF). Animals of the control group received the vehicle only. Additional 5 animals per sex were included into the control and high dose groups and observed for further 14 days subsequently to the 28-day exposure period (recovery groups). No test item-related mortality occurred throughout the study period. Clinical signs associated with local gastric irritation of the test item were histopathologically confirmed and were related to the specific administration route used in this study and therefore considered not relevant for the human risk assessment. There were no treatment-related effects on body weight development and food consumption noted throughout the study period. There were no test item-related effects noted at ophthalmoscopic evaluation and on haematology, clinical chemistry, urinalysis and neurobehaviour. Gross pathology revealed no test material-related changes. Kidney weights were found to be statistically significantly increased (absolute weight 16%, relative weight 15% above controls) in high dose males (600 mg/kg bw/day); this was associated with histopathological findings in the kidneys of these animals. In fact, minimal to moderate multifocal tubular degeneration/regeneration was observed in the kidneys of all high dose group males and females (600 mg/kg bw/day), which in males further was accompanied by minimal tubular dilation. Moreover, the kidneys in most animals showed crystalline deposits within altered tubules. In a single female of this dose group, a minimal focally extensive transitional cell hyperplasia was seen in the renal pelvis. In the urinary bladder focally extensive transitional cell hyperplasia was also noted in two animals of the high dose group (600 mg/kg bw/day).

Renal and urinary bladder changes noted in high dose animals (600 mg/kg bw/day) might be due to the oxalate content of the test item. Crystals found in kidneys of males could be renal calculi developed from calcium oxalate formation and precipitation. Oxalate toxicity is known to be both dose- and sex-dependent (Khan, 1997), which explains, why effects noted in this study are restricted to the high dose group and are more severe in males than in females. As the effects observed were not reversible after the 14-day recovery period they are considered to be adverse. The biological behaviour of oxalate is considered to be almost identical between rats and humans (Khan, 1997), thus, the effects noted in this study are considered relevant for the human risk assessment.

Based on the outcome of this study, the LOAEL was concluded to be 600 mg/kg bw/day for both male and female rats. Consequently, the NOAEL was set at 250 mg/kg bw/day for males and females.

In addition, a reliable key repeated dose rat toxicity study with Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate was conducted for the oral route, according to OECD test guideline 408 (subchronic exposure duration) including fertility parameters, and in compliance with GLP (CBMM Europe BV, Key, 2020, RDT). Ten Wistar rats per sex per dose received daily oral gavages of the test item dissolved in water at doses of 50, 150 and 450 mg/kg bw/day for 90 consecutive days. Analysis of dose formulations revealed that achieved concentrations at week 5 and 13 (last study week) were significantly below the target concentrations. Subsequent investigations revealed that the test item degrades in contact with oxygen. Hence, actually achieved concentrations were recalculated based on the lowest mean values achieved for each dose level at the different testing occasions. Thus, the reevaluated achieved test doses were: 32 mg/kg bw/day, 103 mg/kg bw/day and 311 mg/kg bw/day. Animals of the control group received the vehicle only. The doses selected were based on the results of a 14-day range-finding study (CBMM Europe BV, Supporting, 2013, DRF) and of the 28-day study (CBMM Europe BV, Key, 2014, RDT). No treatment-related mortalities occurred in the control or any of the dose groups during the treatment period of this study. High dose males exhibited statistical significantly lower body weights compared to controls on study day 43 (-8.28%) and 50 (-7.63%). These findings are considered as test item-related, and toxicologically relevant. In accordance with these findings, high dose males exhibited a statistically significantly reduced body weight gain (91.25 g) compared to controls (120.10 g) over the whole study period. No such effects were seen in females. Increased salivation in 1/20 low dose animals, 4/20 mid dose animals and in 15/20 high dose animals as well as moving the bedding in 13/20 high dose animals are deemed to be local reactions to the test item. Spontaneous activity (males: -7.5%; females: -22.50%) and urination (females: -72.73%) were statistically significantly reduced in high dose animals compared to corresponding controls and it cannot fully be excluded that these reductions are correlated to the test item. Apart from that, no other test item-related or toxicologically relevant clinical signs were recorded during the study. No toxicologically relevant effects on food consumption or fertility parameters such as estrous cycle, sperm count, sperm motility and sperm morphology were observed in the control or any of the other dose groups during the entire study period. Besides, no test item-related effects on thyroid hormones, blood coagulation or parameters of clinical biochemistry were determined in any of the other dose groups. Platelet counts were statistically significantly increased in high dose animals (males: +18.18%; females: +17.41%) compared to control animals. It cannot fully be excluded that this effect is test item-related, it is however not considered toxicologically relevant due to the small size of the change and because all other haematological parameters were inconspicuous, i.e., without effects. In mid dose and high dose males as well as in high dose females the urinary pH was slightly lower than in controls (mean of 6.2 vs. 7.1). Moreover, 1/8 mid dose, 2/7 high dose males and 4/10 high dose females exhibited elevated urinary glucose levels (20 mg/dL) compared to the animals of the control and low dose group. Erythrocytes were increased in 4/10 high dose males. The amount of protein was also marginally increased in high dose animals compared to corresponding controls. An abnormal granular surface and / or a pale colour of the kidneys was recorded in 7/10 high dose males and 4/10 high dose females. Mean kidney weight, kidney to body weight and kidney to brain weight ratios were increased in high dose animals (organ weight: 3.0614 g, organ / body: 0.7102%, organ / brain: 145.5157%) compared to controls (organ weight: 2.5835 g, organ / body: 0.5714%, organ / brain: 117.6647%). These findings are in accordance with histopathological findings in this organ and are considered to be test item-related. In the high dose group, treatment with Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate led to transitional cell hyperplasia of the urinary bladder epithelium and renal changes consisting of intratubular deposition of pale yellow, translucent, angular, anisotropic crystals (due to the nature of the test item most probably calcium oxalate crystals) with subsequent nephropathy. These results coincide with the effects found in the 28-Day study and as already discussed there are considered relevant for the human risk assessment. Thus, on the basis of the present 90-Day Repeated Dose Oral Toxicity study with actually achieved dose levels of 32, 103, and 311 mg/kg bw/day the following conclusions can be made:

Under the conditions of this study the no-observed adverse effect level (NOAEL) is considered to be 103 mg/kg bw/day based on reduced body weight gain in males and urinary tract findings noted in males and females at 311 mg/kg bw/day.

References:

Khan, S.R. (1997). Animal models of kidney stone formation: an analysis. World J Urol. 15(4):236-43.

 

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on these data, Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate does not meet the criteria to be classified for specific target organ toxicity after repeated exposure according to EC/1272/2008.