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Administrative data

Description of key information

Repeated dose 90-day oral toxicity study (OECD 408, read-across structural analogue):


NOAEL for systemic toxicity, rats: 600 mg/gk bw/d

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
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)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0001021592
- Expiration date of the lot/batch: December 11th, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stable

The test solution contains NaOH due to the production method.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany

Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 6-7 weeks old
- Weight at study initiation:
males: 177 – 205 g (mean: 190.43 g, ± 20% = 152.34 – 228.51 g)
females: 132 – 155 g (mean: 146.53 g, ± 20% = 117.22 – 175.83 g)

- Housing: - Full barrier in an air-conditioned room
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
- Diet (e.g. ad libitum): - Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): - Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)

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

IN-LIFE DATES: From: February 25th 2016 (study initiation date) To: June 9th 2016 (experimental completion date)
Experimental Starting Date: March 2nd 2016
Treatment Period: March 8th 2016 to June 8th 2016
Route of administration:
oral: gavage
Details on route of administration:
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured and administered at approximately the same time each day.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were used not more than 10 days after preparation.
The test item was dissolved in aqua ad iniectabilia and administered daily during a 90-day treatment period to male and female animals by oral gavage.

For the preparation of test item formulations, the required amount of 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. The mixture was stirred continuously for ca. 1 minute, the amount of test item required to achieve the targeted dose concentration of active ingredient within the formulation sample was calculated based on the concentration of active ingredient within the test item (51.0%).

VEHICLE
Name: aqua ad iniectabilia
Manufacturer: AlleMan Pharma
Batch No.: 503424
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: 02/2018
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the 1st, 5th and 13th 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 (in total 18 samples). For determination of the concentration of test item in dosing formulations, samples from all dose groups were retained in week 1, 5, 9 and 13 of the treatment period. Hence, in total 16 samples were taken for determination of concentration. Each sample was retained in duplicate (sample A and B) with a volume of approx. 5 mL. All samples were stored between -15 °C and -35 °C. The ‘A’ samples were analysed. The samples ‘B’ were retained as back-up until the analyses of samples ‘A’ have been performed.
Concentration results were considered acceptable as sample concentration results were within or equal to ± 10% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤10% for each group.
All samples of dosing formulations were analysed in accordance with GLP under the study reference number 156098. All samples were discarded after completion of the final study report.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group, referred to as Control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low dose, referred to as LD
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Medium dose, referred to as MD
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
High dose, referred to as HD
No. of animals per sex per dose:
10 male and 10 female animals per group
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days.
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 (based on information from previous studies). The health condition of the each animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed observations with focus on 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 were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and during the last week of the treatment period.

Functional Observations
Once before the first exposure and once in the last week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals .
The procedure applied to each animal was briefly as follows: After observing the animal in the home cage, it was transferred to the open field and watched for 2 minutes. Thereafter animal was removed from the arena and manipulated to assess reflexes, grip strength, pupil response and reactions to handling were tested and approx. 10 minutes after returning the animal to the home cage, body temperature was measured rectally. During the length performance of this procedure data of on a battery of 47 motor-behavioural, visceral and sensory parameters were assessed and collected.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Not specified
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined in blood collected at the end of the treatment prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Intraperitoneal injection of ketamine and xylazin solution
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animal
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table 5 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 6 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 8)
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period following blood sampling were sacrificed using anesthesia (by injection of ketamine and xylazin solution into the inferior vena cava) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities
Organ Weight
The wet weight of the selected organs (seen in Table 8) from all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not taken.

HISTOPATHOLOGY: Yes (see table 9)
The tissues (in Table 9) from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
Statistics:
Evaluation of Results and Statistical Analysis:
The findings of this study were evaluated in terms of the observed effects.
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. Mean group body weights were calculated.
The individual and mean group 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 (as group mean in summary tables and/or listed in individual data tables) (Annex 1 in the full study report).
Analytical results and histopathological findings are presented in separate phase-reports attached to thhe report (Annex 2 and Annex 3 in the full study report).
With few exceptions, toxicology and pathology data were captured, using the validated departmental computerized system Ascentos® System (version 1.1., Pathology Data Systems Ltd.). Otherwise, raw data were recorded on paper according to appropriate SOPs.
A statistical assessment of the data on body weights, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values from dosed animals with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical analyses were performed with Ascentos 1.1.3 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):
There were no treatment-related adverse observations in either sex.
The following clinical findings were recorded during the study:
Salivation and moving the bedding were observed in MD and HD animals, immediately after administration with a roughly similar incidence among those groups. These clinical findings, closely related to the administration procedure, indicate a local effect of the test item formulation and are not considered as signs of adverse systemic effects. Similar findings were not observed in males and females of the LD and C groups.
In addition, on one single day in one female and on two occasions in another female, both from the HD group, abnormal breathing was recorded after dosing. Alopecia was also observed in 2 females of LD group for 6 days. As these effects were observed only on few days and/or with no dose response, they are considered not to be toxicologically relevant.
With the exception of moribund condition of animal no 32 (male HD,) no other clinical observations were recorded during the daily observations.
During the course of the study, the following parameters of the weekly detailed clinical observations were statistically different to the control in males: salivation higher in HD in week 6 (HD 4.7, control 4.0), in week 7 (HD 4.9, control 4.0), in week 8 (HD 6.0, control 4.0), in week 9 (HD 4.9, control 3.7), in week 10 (HD 4.9, control 4.0) and in week 11 (HD 5.1, control 4.0), spontaneous activity lower in HD in week 7 (HD 3.6, control 4.0), in week 8 (MD 3.2, HD 2.6, control 3.9) and in week 9 (HD 3.6, control 4.0). Furthermore, in week 3, lower animal sleeps score in MD and HD (MD 0.0, HD 0.0, control 0.5) and higher animal moving in the cage score in MD and HD (MD 1.0, HD 1.0, control 0.5) was observed.
The following parameters of the weekly detailed clinical observations were statistically different to the control in females: salivation higher in HD in week 4 and 5 (HD 4.6, control 4.0), in week 7 (HD 5.0, control 4.0), in week 8 (HD 5.2, control 4.0), in week 9 (HD 6.0, control 4.0) and in week 10 (HD 5.4, control 4.0), spontaneous activity lower in MD and HD in week 9 (MD 3.4, HD 3.2, control 4.0) and in week 10 (HD 3.5, control 4.0). Furthermore, in week 8, lower animal sleeps score in LD, MD and HD (LD 0.0, MD 0.0, HD 0.0, control 0.3) and higher animal moving in the cage score in LD, MD and HD (LD 1.0, MD 1.0, HD 1.0, control 0.7) was observed.
All other detailed clinical observation parameters were within the normal range of variation of historical control data for this strain. Any statistically significant differences between dose and control groups are not assumed to be biologically relevant.
There were no ophthalmoscopic findings in any of the animals of this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no treatment-related mortality in either sex.
Animals no. 31 (male HD) was found dead on day 84 and male no. 32 (male HD) was euthanized for ethical reasons on day 35.
Animal no. 70 (female MD) was found dead on day 12 but was almost completely cannibalised by the cage mates and therefore no tissues were available for histopathology.
Animal no. 32 was euthanized in a moribund condition. Until this condition occurred clinical signs like abnormal breathing, salivation, piloerection and hunched posture had been observed for a week. Although the gross lesions recorded in animals nos. 31 and 32 (affections of the thoracic cavity) were indicating a gavage error, the histological evaluation of the organs and tissues did not reveal the cause of morbidity/death of these animals.
The cause of death of the animal no. 70 could not be established as no tissues were available for histopathology.
Apart from that no mortality occurred in the control or any of the dose groups during the treatment and recovery period of this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In both males and females, the mean body weight increased with the progress of the study in all groups.
In males, the mean body weight was 10-11 % higher in the HD group from day 36-90 without achieving statistical significance. On day 90 the difference in body weight of the HD males to controls reached 11.02 %. This body weight increase is considered to be a test item related. Moreover increase in MD male body weight was also observed but this increase was marginal (0-4.68 %).
In females, the mean body weight was 5-8 % higher in the HD group from day 36-90 and statistical significance was achieved on day 50 and 64 when compared with the controls. This mean body weight was within the expected historical range of variation with no considerable differences between dose groups and control group during the treatment period.
In HD males the overall body weight change from day 1 to 90 was higher without achieving statistical significance compared to the controls. However, statistically significantly higher body weight change was observed during day 85-90 in MD and HD groups when compared with the controls.
In accordance with body weight development, in females, the weight gain was within the expected historical range of variation. However, overall body weight change from day 1 to 90 was marginally but statistically significantly higher in MD and HD group when compared with the controls.
Therefore these finding in HD males but not females represent test item relation. However, toxicological significance of this increase in body weight gain in males was not clear.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In correlation to body weight gain, the food consumption in males and females was higher at the beginning of the study and then constantly decreased with the progress of the study in all groups, which is considered to be a normal development as food demand is usually highest at a young age and then constantly decreases. During the whole treatment period, food consumption of the male and female HD group was higher compared to the corresponding control group, which correlates to the higher mean body weight and body weight gain of the male and females in these groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In female, statistically significantly higher WBC (MD 3.436, HD 3.750 and control 2.065) and PLT (MD 851.8, HD 855.6 and control 697.5) counts were observed in MD and HD group when compared with the controls. These statistically significant differences between dose and control groups in females are not assumed to be biologically relevant as no such effect was observed on differential WBC count and values for both WBC and PLT were within the normal range of variation of historical control data.
All other haematology parameters in males and females were within the normal range of variation of historical control data for this strain.
Blood coagulation was not affected by IBP1-Na. The observed values were within the normal range of variation of historical control data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the female, statistically significantly lower ALAT (17.97 % of control) and ASAT (20.31 of control) were observed in HD group when compared with the controls. These statistically significant differences between dose and control groups in females are not assumed to be biologically relevant as decrease in these liver enzymes have no biological significance and values for both ALAT and ASAT were within the normal range of variation of historical control data.
Besides, all parameters of clinical chemistry in males and females were within the normal range of variation of historical control data for this strain and are not assumed to be toxicologically relevant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Slightly high protein and erythrocytes levels were found in the urine of few male and females of all groups including controls. As this effect was observed in animals of all groups including control, it was considered to be isolated findings with no toxicological relevance.
All other parameters of urinalysis in test item treated males and females at the end of the treatment period were not considerably different to the corresponding control and were within the normal range of variation of historical data for this strain.
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period in HD group of both, males and females, a moderate statistically significantly higher mean absolute and relative (to body weight) weight of liver was recorded, compared to the corresponding control (mean absolute/relative +29%/+18% in males and +13%/+9% in females). In histology, this effect was associated with centrilobular hepatocellular hypothrophy in both genders. There were no further indicators for toxicity in any of the organs and tissues examined. Therefore, this finding is not considered to be of adverse nature, but related to increase in liver metabolism.
The kidney of HD males but not females was statistically significantly higher with mean absolute organ weight value of 21% above the C group. Histopathologically, there were minor changes characterized by minimal tubular dilation associated with a swelling (vacuolation) of the epitehlia and such lesions are in general reversible. There were no findings indicating a morphological injury (see also histopathology phase report) and therefore this effect on male kidney weight was not considered to be adverse.
In HD males, a statistically significantly higher absolute mean weight of prostate with seminal vesicles and coagulation glands (3.38 g in HD vs 2.80 g in C) was recorded. In the light of the absence of histopathological effect, this effect on prostate with seminal vesicles and coagulation glands weight was not considered to be adverse.
Besides, all male and female organ weights were within the normal range of variation of historical control data for this strain and differences between dose and control groups are not assumed to be biologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pathological changes were observed in the following animals. Abnormal red color and fluid filled thoracic cavity was observed in male no. 31 (HD). The thymus showed abnormal dark color in male no. 32 (HD). There was also black focus on lung and gas filled stomach and gastrointestinal tract was observed in male no. 32. Histopathologically, lung finding was due to congestion and for stomach finding, no histopathological correlate was observed.
The spleen of female no. 66 (MD) was reported to be enlarged. In histology, this finding correlates with congestion. There was also a cyst on ovary found in female no. 55 of LD group for which no histopathological correlate was observed.
Fluid filled uterus with cervix was recorded in 2/10 LD females (no. 51 and 54), 2/10 MD females (no. 63 and 65) and 2/10 HD females (no. 71 and 80). In histology, this finding correlates with female cyclic change and represents a normal background finding for this strain.
Female no. 70 could not be observed for macropscopic findings as the animal was completely cannibalised by the cage mates.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Under the conditions of this study, there were three decedents at 600 mg/kg. The cause of morbidity/death could not be established.
Gross lesions were not remarkable, however, histopathology revealed lesions in the liver and kidneys (males only) at a minor severity. Secondary lesions were noted in the adrenal glands and thymus.
In the liver, there was hepatocellular hypertrophy (mainly centrilobular) at minor severity degrees at 600 mg/kg in both sexes. There were no further indicators for toxicity in any of the organs and tissues examined. Therefore, this finding is not considered to be of adverse nature, but related to increase liver metabolism.
In male kidneys at 600 mg/kg, there were minor changes characterized by minimal tubular dilation associated with a swelling (vacuolation) of the epitehlia, mainly in the rectal and distal tubules. The nature of this lesion is unknown but is likely related to storage in tubular cells and subsequent water uptake/disturbed release. Such lesions are in general reversible. There were no findings indicating a morphological injury.
Secondary findings, deemed to be stress-related consisted of diffuse cortical hypertrophy (zona fasciculata) at a minor severity in both sexes at 600 mg/kg, and a minimally increased thymic atrophy in males only.
Indication for stomach irritation (Lymphoid follicles, Incr.inflamm.infilt., vacuolation and inflammation) was noted in the high dose females and to a less extend at high dose males

In general, these findings are minor in severity and not considered to be adverse.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with IBP1-Na in male and female Wistar rats, with dose levels of 30, 150, and 600 mg/kg body weight/ day the following conclusions can be made:
No effect of toxicological relevance was observed on male and female body weight, food consumption, haematology, clinical biochemistry, urine parameters, clinical signs, gross pathology and organ weight up to 600 mg/kg bw/day.
Indication for stomach irritation (Lymphoid follicles, Incr.inflamm.infilt., vacuolation and inflammation) was noted in the high dose females and to a less extend at high dose males.
The histomorphological no-observed-adverse effect level (NOAEL) was established at 600 mg/kg bw/day in males and female rats.
Based on the study findings, the overall NOAEL of IBP1-Na for systemic toxicity in this study is considered to be at 600 mg/kg bw/day.

Remark: The test substance as produced and marketed contains NaOH due to the production method.
Executive summary:

The substance was investigated in a 90-Day Repeated Dose Oral Toxicity Study according to OECD Guideline 408 (adopted 21 September 1998). Doses of 0 (control), 30, 150 and 600 mg/kg bw/day was administered by gavage daily over a period of 90 days to 10 Wistar rats/sex/dose.


No effect of toxicological relevance was observed on male and female body weight, food consumption, hematology, clinical biochemistry, urine parameters, clinical signs, gross pathology and organ weight up to 600 mg/kg bw/day. Indication for stomach irritation (Lymphoid follicles, Incr.inflamm.infilt., vacuolation and inflammation) was noted in the high dose females and to a less extend at high dose males.
The histomorphological no-observed-adverse effect level (NOAEL) was established at 600 mg/kg bw/day in males and female rats.


Based on the study findings, the overall NOAEL of IBP1-Na for systemic toxicity in this study is considered to be at 600 mg/kg bw/day.


 


 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification