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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 7-8 weeks old, females: 7-8 weeks old.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 55 +- 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: From: 03 December 2012 To: 08 April 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad injectionem
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 10 mg/ml, 60 mg/ml, 160 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required): Manufacturer: AlleMan Pharma; Batch No.: 2621051-2
- Purity: 100 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the concentration verification samples were retained from all groups in the first week, at the initiation of month 2 and 3 and in the last week
stored between -15° and -35° C (16 samples in total). Stability of the dosing formulations was not tested. In the first week of treatment and at
the initiation of month 2 as well as end of month 3 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 (18 samples in total). All samples were stored frozen
(approximately -20°C) until the analysis was performed. The determination of test item concentration in the dosing formulations was
performed by the analytics department of BSL Bioservice in accordance with GLP. The procedures followed were described in a detailed
analytical phase plan
Duration of treatment / exposure:
Period of 90 days
Frequency of treatment:
Once a day; 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
50 mg/kg body weight; 300 mg/kg body weight; 800 mg/kg body weight;
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period). 5 animals per gender
of the C and of the HD group were subjected to necropsy 28 days after the last administration (end of recovery period).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): 1 animal per group after each other

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter.

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: Yes
Weekly during the treatment and recovery period

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 as well as at the end of the
recovery period in the recovery animals.
- Dose groups that were examined: all animals of all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal sacrifice
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: Yes
- How many animals: All animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal sacrifice
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: at terminal sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once in the last week of exposure as well as in the last week of the
recovery period
- Dose groups that were examined: all animals of all dosing groups
- Battery of functions tested: multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals of all groups were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents
HISTOPATHOLOGY: Yes
A full histopathology was also carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for treatment-related changes that are observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. These examinations were also extended to animals subjected to necropsy at the end of the recovery period.
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.6.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item related clinical signs were observed in MD and HD animals
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item related clinical signs were observed in MD and HD animals
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:
effects observed, treatment-related
Description (incidence and severity):
Some parameters were affected but none of them is assumed to have any toxicological relevance
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):
Some ogan weights are affected and a weight loss of the hearts (at the end of recovery period; HD) cannot be excluded to be test item related.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
Three males (No. 37, 47 and 50) and five females (Nos. 82, 84, 87, 97 and 99) treated at 800 mg/kg/day did not survive the treatment period.
Hence 3/15 male HD animals and 5/15 female HD animals were concerned.
In histopathology, the deaths of males No. 37, 47, 50 as well as female No. 82 were considered to be due to a local irritant effect of the test item
formulation, when accidentally reaching the airways - most probably due to regurgitation followed by aspiration
Furthermore, histopathology revealed for female No. 99 an extensive ulcerative skin lesion. This was associated with reactive hyperplasia of the
axillary lymph node and minimally increased myeloid:erythroid ratio of the bone marrow.
Hence, for animal Nos. 37, 47, 50, 82, and 99 a systemic test item related effect cannot be stated.
Within females No. 84, 87 and 97 the direct cause of death could not be determined by histopathological evaluation. Atrophy of lymphoid organs was seen in all three and was considered to be due to the bad terminal condition of these animals. All three animals exhibited multiple clinical symptoms. Since the cause of death could not be explained by histopathology or by any other parameter, a systemic test item relation is possible and
cannot be excluded.
.

Clinical Observations
Male and female MD and HD animals showed test item-related clinical signs. Predominant clinical signs observed in male animals during the
application period were slight piloerection (15/15 HD animals, 3/10 MD animals), red nasal discharge (3/15 HD animals), abnormal breathing (
10/15 HD animals), diarrhoea (13/15 HD animals), moving the bedding (13/15 HD animals), slight salivation (12/15 HD animals, 6/10 MD animals)
and severe salivation (4/15 HD animals).
During the recovery period the most prominent symptom was diarrhoea (2/5 HD animals). Other clinical symptoms were found individually
during the recovery period in male animals.
Predominant clinical signs observed in female animals during the application period were abnormal breathing (13/15 HD animals), slight piloerection (14/15 HD animals, 2/10 MD animals), moderate piloerection (7/15 HD animals), moving the bedding (14/15 HD animals, 3 /10 MD animals), slight salivation (10/15 HD animals, 5/10 MD animals), moderate salivation (7/15 HD animals, 1/10 MD animals) and red nasal discharge (4/15 HD animals, 3/10 MD animals).
During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no
ophthalmoscopic findings in any of the animals of this study.


Functional Observation Battery
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.

Body Weight Development
In both males and females, the mean body weight increased with the progress of the study in all groups.
A tendency towards an attenuated body weight gain was observed in male animals of the high dose group during the second half of the
treatment period. Since this was not statistically significant it is not assumed to be test item related and of toxicological relevance.

Food Consumption
In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the
study in all groups.
No considerable effect of the test item on food consumption was found in any of the treatment groups of male animals and in females.

Haematology and Blood Coagulation
At the end of the treatment period a statistical significant increased amount of white blood cells was measured in MD group (+45 %) when compared to C group (p<0.01). However, a dose dependency was not observed. Hence, no test item relation is assumed.
Besides, all haematological parameters were within the normal range of variation no further statistical significance was observed – at the end
of the application period or the recovery period.
Blood coagulation was not affected by the test item at the end of the application period or recovery period.

Clinical Biochemistry
In male animals, values of AP were slightly and dose dependently decreased in treatment groups when compared to C group.
In particular 94.7 U/L (C), 82.6 U/L (LD), 75.9 U/L (MD), and 71.8 U/L (HD) were measured. Furthermore, a slight decrease was also mentioned
for HD group (74.67 U/L) when compared to C group (83.60 U/L) at the end of the recovery period. These differences were not statistical significant between the treatment groups and the C group. Furthermore, the measured differences were just little and still in the range of our historical
control data. Hence, a toxicological relevance is not assumed.
TP was significantly (p<0.01) decreased in male HD animals at the end of the treatment period when compared to C group. Respective 58.74 g/L (C) and 53.99 g/l (HD) were observed. No difference was detected between C and HD animals at the end of the recovery period. This above mentioned
slight difference has no biological and toxicological relevance.
In male animals TBA was dose dependently increased in treatment groups when compared to C group. In particular 32.19 (C), 41.83 (LD), 48.98 (MD), and 62.40 (HD) were measured. No change was found between C and HD group at the end of the recovery period. Except the HD group (at the end of the application period), the values are in the range of our historical control data. The HD value is slightly above the upper limit of our historical
control data. An increase in TBA may indicate the liver as a potential target organ. Blood levels of bile acids may be increased as a result of
diminished hepatic elimination. However, this can be excluded within this study. Neither organ weights, nor histopathology nor any other liver
marker of biochemistry confirms the liver as a target organ. Furthermore, the increase was not statistical significant and the HD value was just
slightly higher than the upper limit of our historical control data. Hence, this increase is not assumed to be of toxicological relevance within the
progress of this study.
In male recovery animals, amount of cholesterol was significantly increased in recovery HD group (1.06 mmol/L) when compared to recovery C
animals (0-82 mmol/L). Since both values are in the range of our historical control data, this difference is not assumed to be biologically relevant.
In female animals, a slight but statistical significant (p<0.05) increase was detected for Na and K of HD animals at the end of the recovery period.
In particular, 149.40 mmol/L Na (C), 152.67 mmol/L Na (HD), 3.63 mmol/L K (C), and 4.81 mmol/L (HD) were measured. These differences were not observed at the end of the treatment period. The K-Value for the HD group was slightly above to what could be expected taken into consideration
our historical control data. Increased K might indicate a disturbance of the kidneys. However, no other parameters (e.g. histopathology or urine)
clearly indicate the kidneys as target organs. Hence, it is not assumed that this increase in K has any toxicological relevance within this study.
Besides, all parameters of clinical chemistry were within the normal range of variation for this strain.


Urinalysis
Moderately increased protein levels were found in male HD animals No. 31, 35, 36, and 39. These increased protein levels could not be found
at the end of the recovery period. Since these increased protein levels were also not seen in female animals and since no histopathological changes
were found in kidneys it is not assumed that these increases are treatment related and of toxicological relevance.
In female animals, no change in any of the urine parameters could be observed at the end of the treatment or the recovery period.

Pathology
Few specific gross pathological changes were recorded for the male and female animals which were not considered to be treatment-related.
Only a gased gastro-intestinal tract could be found for individual animals of the HD group which could be explained by a direct irritant effect of
the test item in the GIT.

Organ Weight
In males, all absolute organ weights were comparable between the treatment groups and the control group at the end of the treatment period.
There were no statistical significant or biological relevant differences. This was confirmed by relative organ weights (to brain weight and to body
weight).
At the end of the recovery period, no statistical significant change was found regarding organ weights when compared male treatment groups with
the C group. However, adrenals were non-significantly increased in male HD group concerning absolute weights (+27 %) as well as relative weight
(in relation to brain weight: +21 %; in relation to body weight: +15 %).
Stress is able to cause hyperplasia and hypertrophy of the adrenal gland due to the hypothalamic-pituitary-adrenal axis and/or the sympatho-adrenomedullary system [9]. However, no systematic hyperplasia or hypertrophy could be found during histological analysis. Hence, a test item relation
of the increased adrenal weights is possible, but due to the missing results in histopathology, due to the missing change at the end of the treatment period as well as due to the limited number of observed animals (3/5 due mortalities before the end of the recovery period) not probable. It is not assumed that this increase has any toxicological relevance. 
In female animals, no statistical significance was found when comparing organ weights (absolute and relative) of treated groups with the C group
at the end of the treatment period. Furthermore, except the uteri (with cervix) no biologically relevant change was found. Absolute weights of uteri
(with cervix) were moderately decreased in treated groups at the end of the treatment period. In particular, -29% (LD), -13 % (MD), and -28% (HD)
were measured. This decrease could be confirmed by relative uteri weights (to body weight and to brain weight).
These weight decreases were still present at the end of the recovery period. In particular, -19 % (absolute), -13 % (relative to brain weight), and -17%
(relative to body weight) were calculated/measured. A statistical significance as well as a dose dependency is missing. Hence, a test item relation
is not assumed.
Absolute weights of hearts were significantly decreased (-12%) in HD group at the end of the recovery period. This could be confirmed by relative
heart weights (to brain weight; p<0.01; -16%). The slight decrease (-11%) for relative heart weight (to body weight) was not statistically significant.
A test item relation is possible but seems unlikely since no histopathological change was observed. The toxicological relevance is not clear.
Beside these described weights, all other organ weights were comparable among the groups and no statistically significant or biological relevant
difference was measured.


Histopathology
Three males and five females treated at 800 mg/kg/day did not survive the treatment period.
Premature death of three of them was clearly attributable and death of another rat was possibly attributable to a local irritant effect of the test item
formulation, when accidentally reaching the airways, e.g. via misgavaging or regurgitation. Moribund condition of one further rat was due to an
incidental focally extensive dermatitis. For the remaining three decedents, a clear cause of death could not be determined by extensive histopathological evaluation, and a direct test item relationship can therefore not be fully excluded. Also, the cause of gaseous distension of the gastrointestinal
tract in one found dead and three prematurely sacrificed animals could not be established, although it may partially be related to the agonal state
of these animals.
At terminal sacrifice, no histological changes of toxicological relevance were seen. Some histopathological lesions in the trachea, lung and stomach were considered to be due to a local irritant effect of the test item formulation and therefore to be without toxicological relevance for humans. In the trachea, minor lesions were seen in occasional animals of all dose groups. Lesions in the lung and stomach were restricted to the 800 mg/kg/day dose group. All of these were completely reversible after the recovery period.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality

Target system / organ toxicity

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

Applicant's summary and conclusion

Conclusions:
On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with the test item in male and female Wistar rats, with dose
levels of 50, 300, and 800 mg/kg body weight day the following conclusions can be made:
At a dose level of 800 mg/kg body weight clinical symptoms occurred in more animals than in control animals. Furthermore, the reason for 3 animals (deaths before terminal sacrifice) could not be determined. Hence, a test item relation cannot be excluded.
At a dose level of 300 mg/kg body weight clinical symptoms occurred in more animals than in control animals. However, these symptoms were signs of discomfort and not of direct toxicological relevance. Hence, the NOAEL in this study is considered to be 300 mg/kg body weight
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test item via oral administration to rats over a period of 90 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised of 10 male and 10 femaleWistarrats(5 male and 5 female animals per recovery group).

During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery group were observed for a period of 28 days following the last administration.

Body weight and food consumption were measured weekly. At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

A full histopathological evaluation of the tissues was performed on high dose and control animals. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. These examinations were also extended to animals subjected to necropsy at the end of the recovery period.Any gross lesion macroscopically identified will be examined microscopically in all animals.

Any gross lesion macroscopically identified will be examined microscopically in all animals.

The following doses were evaluated:

Control:                       0         mg/kg body weight

Low Dose:                   50       mg/kg body weight

Medium Dose:             300    mg/kg body weight

High Dose:                  800    mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad injectionem and administered daily during a 90-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.


 

Summary Results

Three males (No. 37, 47 and 50) and five females (Nos. 82, 84, 87, 97 and 99) treated at 800 mg/kg/day did not survive the treatment period. A systemic test item relation can be excluded for five of the above mentioned animals. For 3 animals the exact cause of death cannot be determined and hence a systemic test item relation is possible.

Male and female MD and HD animals showed test item-related clinical signs.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

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

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.

In both males and females, the mean body weight increased with the progress of the study in all groups.

No considerable effect of the test item on food consumption was found in any of the treatment groups of male animals and in females.

No test item related change was found for haematological parameters.

Blood coagulation was not affected by the test item at the end of the application period or recovery period.

In male animals, values of AP were slightly and dose dependently decreased in treatment groups when compared to C group. A toxicological relevance is not assumed.

TP was significantly (p<0.01) decreased in male HD animals at the end of the treatment period when compared to C group. This difference has no biological and toxicological relevance.

In male animals TBA was dose dependently increased in treatment groups when compared to C group. Except the HD group (at the end of the application period), the values are in the range of our historical control data. All changes (including the change in HD group) are not assumed to be of toxicological relevance.

In female animals, a slight but statistical significant (p<0.05) increase was detected for Na and K of HD animals at the end of the recovery period. It is not assumed that these increases have any toxicological relevance within this study.

Moderately increased protein levels were found in urine of male HD animals No. 31, 35, 36, and 39. These increases are not assumed to be treatment related and of toxicological relevance.

Few specific gross pathological changes were recorded for the male and female animals which were not considered to be treatment-related.

Adrenals were non-significantly increased in male HD group concerning absolute weights (+27 %) as well as relative weight (in relation to brain weight: +21 %; in relation to body weight: +15 %). It is not assumed that these increases have any toxicological relevance.

Absolute weights of uteri (with cervix) were moderately decreased in treated groups at the end of the treatment period. A test item relation is not assumed.

Absolute weights of hearts were significantly decreased (-12%) in HD group at the end of the recovery period. This could be confirmed by relative heart weights (to brain weight; p<0.01; -16%). A toxicological relevance is not clear but seems unlikely since no histopathological change was observed.

At terminal sacrifice, no histological changes of toxicological relevance were seen in the animals surviving the end of the treatment or recovery period.

Conclusion

On the basis of the present study with dose levels of 50, 300, and 800 mg/kg bw/d day the following conclusions can be made:

At a dose level of 800 mg/kg/ bw/d clinical symptoms occurred in more animals than in control animals. Furthermore, the reason for 3 dead animals (deaths before terminal sacrifice) could not be determined. Hence, a test item relation cannot be excluded.

At a dose level of 300 mg/kg bw/d clinical symptoms occurred in more animals than in control animals. However, these symptoms were signs of discomfort and not of direct toxicological relevance. Hence, the NOAEL in this study is considered to be 300 mg/kg bw/d.