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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:
18 April 2012 - 21 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The following parameters were examined for all animals not according to GLP regulations: Cholinesterase, transferring, and transferrin saturation
Limit test:
no

Test material

Constituent 1
Test material form:
other: solid
Details on test material:
Physical State: solid
Colour: black
Storage Conditions: at room temperature, protected from light and stored in closed bags

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: males: 8-9 weeks old, females: 8-9 weeks old
- Weight at study initiation: : males: 150 – 186 g (mean: 169.23 g, ± 20% = 135.39 – 203.08 g); females: 130 - 149 g (mean: 139.38 g, ± 20% = 111.51 – 167.26 g).
- Housing: The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 261111)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Adequate acclimatisation period (at least 5 days)

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

IN-LIFE DATES: From: 23 April 2012 To: 28 August 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad injectionem
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 5 mg/mL, 20 mg/mL, 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): Manufacturer: Diprom; Batch No.: 10952-1
- 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 were tested once at the beginning of the treatment period. From all dose groups samples of control or dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15° and -35 °C (6 samples in total).
In the first week of treatment and at the initiation of month 2 and 3 samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, medium and low dose formulations and stored between -15° and -35 °C (27 samples in total).
At the end of the treatment period all samples of dosing formulations were shipped on dry ice and protected from light to:
CIP GmbH
Chemisches Institut Pforzheim GmbH
Schulberg 17
75175 Pforzheim
Germany
The determination of test mateiral concentration in the dosing formulations was performed by CIP GmbH, in accordance with GLP.
Duration of treatment / exposure:
The test material was orally administered for a period of 90 days
Frequency of treatment:
Once per day; 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
25 mg/kg bw/day; 100 mg/kg bw/day; 250 mg/kg bw/day
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 included in control group and high dose group were observed for 28 days after the last administration (recovery period).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on previuos studies
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): One animal from each group after each other

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (Except weekend and public holidays were the observation was performed once 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: on the first day of administration and weekly during the treatment and recovery period and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined: 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 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 of the animals
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: Yes
- How many animals: All animals of all groups


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 of the animals
- Animals fasted: Yes
- How many animals: all animals of all groups

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment and recovery period
- 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 fourth week of exposure as well as in the last week of the recovery period
- Dose groups that were examined: all animals of all groups
- Battery of functions tested: multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests

Lithium Analysis in Plasma
Li-determination was performed in plasma samples at the end of the treatment period.
After overnight fasting, blood from the abdominal aorta of the animals was collected in tubes containing an anticoagulant (e.g. citrate) and frozen between -15° and -35 °C.
Afterwards, plasma samples of the C animals 1-10 and 51-60 as well as the HD animals 31-40 and 81-90 were shipped on dry ice and protected from light to:

CIP GmbH
Chemisches Institut Pforzheim GmbH
Schulberg 17
75175 Pforzheim
Germany
The determination of Li in the plasma samples were performed by CIP GmbH, in accordance with GLP.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119) were sacrificed using anesthesia (ketamine, medistar Arzneimittelvertrieb, lot no: 00212, expiry date: 03/2014 and xylazin, Serumwerk, lot no. 00711, expiry date: 08/2013) and 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.
The wet weight of the below described organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed separately. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded:
liver, uterus and ovaries, kidneys, thymus, adrenals, heart, testes, spleen, epididymides, brain

HISTOPATHOLOGY: Yes

The following organs were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.

brain (cerebrum, cerebellum and medulla/pons), heart, spinal cord, ovaries (females), eye,uterus with cervix (females), liver, vagina (females), kidneys,testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid / parathyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, bone with bone marrow (sternum), lung and trachea, pituitary gland, mammary glands (females), oesophagus, skin, gross lesions
salivary glands, pancreas, aorta

A full histopathology was 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 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 was examined microscopically.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd. (test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
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 5.01 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):
No substance related death occured. Clinical Symptoms were found in treatment groups
Mortality:
mortality observed, treatment-related
Description (incidence):
No substance related death occured. Clinical Symptoms were found in treatment groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly decreased body weight gain in female HD group during the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly decreased food consumption in HD group without effects on body weights.
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):
Slight changes could be found in HD group, which remained in the range of historic controls or were not statistically significant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Some changes could be found in MD and HD group. Iron concentration and saturation of transfrerrin were increased at the end of treatment.
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 small and inconsistent changes could be found in MD and HD groups. Affected organs were: Adrenals, Liver, Thymus
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Some findings occured in MD and HD groups: Pigmentation of hepatocytes, kidney tubular epithelium, peyer's patches
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
One animal of the MD group (No. 75, female) was euthanized due to animal welfare considerations. However, this animals bad condition is not considered to be test item related since it was mostly related on inflammation of the skin and no other animal exhibited this severe inflammation.

Clinical Observations
The male animals of the main group exhibited individual clinical findings which were not related to the test item. The main female animals exhibited more symptoms in HD group compared to the C group or the other treatment group – e.g. 8/10 HD animals showed moving the bedding, 5/10 HD animals slight piloerection. In the recovery groups (male and female) no clear test item related tendency of increased clinical symptoms was found.
This increased incidence of clinical symptoms in HD female group is assumed to be related to the test item. However, these clinical symptoms are not considered to be adverse and of toxicological relevance since these mostly indicate discomfort of the animals.
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 and recovery 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 the C and treatment groups. Throughout the treatment period, body weights and body weight gain were within the normal range of variation for this strain.
During the recovery period a slight tendency to a decreased body weight was recorded for female HD group. However, this was not statistical significant. Body weight of male HD animals as well as body weight gain of male and female HD animals was comparable to C group. No significant difference was found.
A test item relation is possible. However, due to the mild characteristics and the absent statistical significance this attenuated weight gain has no toxicological relevance.

Food Consumption
Mean food consumption of female HD animals was significantly attenuated in HD group during the whole treatment period (p<0.05). For MD and LD group, no significant change was evaluated when compared to C group. During the recovery period the mean food consumption was still reduced for HD female animals (p<0.01). For male animals during treatment and recovery period no difference to C group could be found.
The decreased food consumption is assumed to be test item related. However, due to the mild characteristics of the decrease and without any significant correlation to a decreased body weight a toxicological relevance cannot be stated.

Haematology and Blood Coagulation
At the end of the treatment period, a higher percentage of neutrophils was found in male HD group (31.42 %) when compared to the C group (24.19 %). The value is still in the range of our historical C data. At the end of the recovery period, the difference in neutrophil percentage was slightly and non-significantly remaining. A very slight increase in the percentage of neutrophils was also observed in the female HD animals at the end of the treatment period (HD: 26.48 %; C: 20.94 %). Again, the value is still in the range of our historical C values. In female recovery HD animals the percentage of neutrophils is strongly decreased (20.7 %) when compared with the C mean value (38.75 %) at the end of the recovery period. However, this belongs to a very high value of C animal No. 91. Taken this female animal out of the calculation, no further difference in the percentage of neutrophils at the end of the recovery period could be observed. Furthermore, due to clotted blood samples, only 2 animals of females in the HD recovery group could be evaluated.
These slight changes in the percentage of neutrophil cells are not clearly assumed to be test item related and are not assumed to be of toxicological relevance – due to the mild characteristics.
No difference could be found for parameters of coagulation in the treatment groups when compared to the C group.

Clinical Biochemistry
At the end of the treatment period, the amount of iron found in serum of male HD group was significantly (p<0.05) increased indicating an absorption of the test item through intestinal barriers. As a consequence, the transferrin saturation was significantly (p<0.05) increased, too. At the end of the recovery period, no further difference could be found regarding iron content of transferring saturation when comparing C or HD group. Again, in female animals amount of iron, found in serum, was significantly increased for HD group.
A significant change could not be mentioned for the transferring saturation of female HD animals which can be explained by relatively high transferring saturation of the main C animals. At the end of the recovery period, all changes found after the treatment period could not be found again indicating clearance via liver or kidneys. The increased amount of iron, due to absorption though intestinal barriers, is not assumed to be test item related.
A toxicological relevance of the increased amount of iron as well as the increased transferring saturation cannot be considered within the duration of this study. In principal, iron deposition may lead e.g. to formation of reactive oxygen species leading to lipid peroxidation, protein oxidation and liver fibrosis. However, within this study neither histopathology nor clinical biochemistry or organ weights revealed toxic effect of the increase amount of iron.
All other parameters of clinical chemistry of male animals were within the normal range of variation for this strain and any slight changes between C and treatment groups – even when statistically significant like Urea in HD group or Glucose in MD group - are not assumed to be of biological relevance.
Values of AP of female animals at the end of the application period were increased in MD and HD group and the difference of MD vs C group was statistically significant. In particular, 36.3 U/L (C), 62.0 U/L (MD), and 49.8 U/L were found. The value of the MD group was slightly above to what could be expected regarding our historical C data. However, since no dose dependency was found, since the HD value is in the range of our historical C data, and since during the recovery period this increased AP amount recovered totally, this increase - although possibly test item related - is not assumed to be of toxicological relevance within this study.
Values of TBA of female animals were increased at the end of the application period for MD (51.17 U/L) and HD (54.06 U/L) group when compared to C group (27.64 U/L). However, the variation within the groups was relatively high and hence no test item related influence could be clearly mentioned.
Besides, all parameters of clinical chemistry of female animals were within the normal range of variation for this strain and statistically significant differences between dose and control groups are not assumed to be biologically relevant.

Urinalysis
High erythrocyte amounts were found in the urine of male animal numbers 76 (MD) and 85 (HD). In this regard, the urinary bladder of animal No. 85 was minimal hemorrhagic. Although this is a direct association, a technical problem due to the necropsy could be also responsible for this. In principal, a test item relation cannot be excluded. Since only 2 animals exhibited this very high blood value in urine, no toxicological relevance due to the test item can be attributed and a technical problem during the necropsy seems probable.
Besides, all urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed.

Pathology
Few specific gross pathological changes were recorded for the male and female animals which were not considered to be treatment-related since the distribution of the findings was similar in C and treatment groups.

Organ Weight
In males, absolute weights of adrenals were slightly increased in MD (+11 %) and HD (+10 %) animals. This could be confirmed for relative adrenal weights (to body weight but not to brain weight). In particular, an increase of 15 % (MD) and 21 % (HD; p<0.05)) could be mentioned. For Recovery HD group, small increases could be mentioned when compared to recovery C group. In particular, 8 % (absolute), 7 % (relative to brain weight), and 4 % (relative to body weight) were found. For female animals, no change was found for adrenal weights at the end of the treatment period. However, at the end of the recovery period adrenal weights of HD female animals were decreased for 11% (absolute), 9 % (in relation to brain weight), and 6 % (in relation to body weight).
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 [10]. This stress related increase of adrenal weights is often associated with a decreased thymus weight which can be also found within this study. However, no systematic hyperplasia or hypertrophy could be found during histological analysis. Hence, a test item relation of the increased adrenal weights is probable, but the direct toxicological relevance of this mild weight increase is not totally clear for this organ.
Relative weights of hearts (to brain weight) of male animals were statistical significantly decreased (-13%) in male HD group. This could not be confirmed for absolute heart weights and relative heart weights (to body weight). Heart weights of HD group were also not changed at the end of the recovery period. No weight change for hearts could be recognized for female animals. Due to the absence of decreased relative heart weights, a test item relation cannot be considered.
Relative weights (to brain weight) of kidneys of male animals were decreased in HD group (-11 %) when compared to C animals. This was not confirmed for absolute weights or relative weights to body weight. Furthermore, at the end of the recovery period no kidney weight change was mentioned for male HD animals. No weight change of kidneys (absolute and relative) could be recognized for female animals at the end of the treatment period. At the end of the recovery period, a significant decrease in absolute kidney weights was recognized for female HD animals (-10 %; p<0.05). Relative organ weights were slightly decreased (-9% in relation to brain weight and -6% in relation to body weight). A test item relationship cannot be excluded. However, due to the mild characteristics of the weight change as well as the inconsistency among the weights/calculated weights (absolute and relative) a toxicological relevance could not be stated within this study.
In male animals, relative weights (to brain weight) of spleens were slightly decreased in MD and HD animals at the end of the treatment period. Respective -9% and -11 % could be measured. This was not confirmed for absolute spleen weights or relative spleen weights and was not found at the end of the recovery period. No change in spleen weights could be found for female animals – neither at the end of the treatment nor at the end of the recovery period. A test item relationship is not assumed.
Brain weights were significantly increased in relation to body weight in male HD animals (+12 %). This was not found for absolute brain weights and at the end of the recovery period. No difference could be found for brain weights of treatment groups when compared with C animals. A test item relation is not assumed.
In male HD animals, liver weights were significantly increased in relation to body weight (+10 %).This was not confirmed in relation to brain weight or for absolute male liver weights. However, at the end of the recovery period an increase of liver weights could be found in male animals. Absolute weights were increased for 10 % (p<0.05), relative liver weights (to brain weight) were increased for 10 % (p<0.01), and relative liver weights (to body weight) were increased for 6 % (p<0.05). In female animals, relative liver weights (to body weight) were significantly decreased in LD group (-9 %) and MD group (- 9%). No decrease could be found for HD group of relative liver weights (to body weight). This pattern could be confirmed for absolute liver weights and in relation to brain weight (without statistical significance). These changed liver weights could be test item related. However, due to the mild characteristics, the absent pathological changes in histopathology as well as the absent parameters in clinical biochemistry a direct toxicological relevance of the changed liver weights cannot be mentioned.
In males, absolute weights of thymus were slightly decreased in MD (-10%) and HD (-%) groups when compared to C group. This was confirmed by relative thymus weights (to brain weight) where a decrease could be mentioned for MD (-17%) as well as HD (-18%). Relative thymus weights to body weights were found to be decreased for 10 % (MD) and 6 % (HD). At the end of the recovery period slight decreases of 6 % (absolute weights), 7 % (relative to brain weight), and 10 % (relative to body weight) were observed. In female animals, thymus weights were moderately decreased at the end of the recovery period but not in treatment groups after the treatment period. In recovery HD group, -23 % (p<0.05) was observed for absolute thymus weights, -21% for thymus weights in relation to brain weight, and -18% in relation to body weight.
These decreased thymus weights may indicate a stress related response as the increased adrenal weights. A test item relation is probable. A direct toxicological relevance of the decreased thymus weights could be possible.
Weights of ovaries of female animals were increased in HD group at the end of the recovery period. In particular, +17% (absolute), +20% (in relation to brain weight), and +23 % (in relation to body weight were mentioned). At the end of the treatment period, no remarkable change was found. The increased at the end of the recovery period could be test item related. However, a statistical significance could not be observed, Hence, the toxicological relevance is unclear.

Uterus weights (weighed together with cervix) were decreased in HD animals at the end of the recovery period. -15% (absolute), -13 % (in relation to brain weight), and – 9% (in relation to body weight) were found. No remarkable change was found at the end of the treatment period. Since no statistical significance could be considered as well as no systematic alteration could be found in histopathology, a toxicological relevance cannot be mentioned.
Except, all other absolute organ weights of male and female animals were all in the range of the normal variation for this strain and no test item related difference could be found when comparing the organ weights of the treatment groups with the organ weights of the C group – neither in main nor in recovery groups.

Histopathology
Histopathological findings clearly attributable to the test item were seen in the liver and kidney, in females only. In the liver, minimal or mild brown pigment in periportal hepatocytes was observed in females treated at 250 mg/kg/day, against a low background incidence in the control group. In one high dose female, brown pigment was also seen in hepatic macrophages. In females treated at 100 mg/kg/day, a test item relationship could not be excluded for a minimally increased incidence of hepatocellular brown pigment. After the recovery period, liver changes had not resolved. In the kidney, minimal amounts of brown pigment were noted in the corticotubular epithelium in a low number of females treated at 100 or 250 mg/kg/day. This change was no longer seen after the recovery period.
For findings noted in the Peyer’s patch and mesenteric lymph node, at terminal sacrifice only, a test item relationship could not be excluded. In the Peyer’s patch, multifocal brown pigment was noted in a low proportion of treated animals of both sexes, without clear dose relationship, but was also seen in one control male after the recovery period. In animals treated at 250 mg/kg/day, a test item relationship could not be excluded for this change. Likewise, in the mesenteric lymph node, an increased incidence and severity of sinus histiocytosis was noted in the males and females treated at 250 mg/kg/day, when compared to the controls, and was considered to be possibly test item-related.
In the lung of one male treated at 250 mg/kg/day, marked multifocal subacute bronchopneumonia with presence of dark brown intrabronchiolar and intraalveolar material was considered to be the consequence of partial instillation or regurgitation/aspiration of test item formulation into the lung and not to be of toxicological relevance.
The finding of brown pigment could be a deposition of iron-containing pigment which could be attributed to the test item. However, also other possibilities appear which could be attributed to iron deposition (e.g. due to hemoglobin) and hence, the nature of the possible iron deposition is not totally clear. Furthermore, no pathological changes could be observed histological and no other indicators of a test item related effect (e.g. liver weight, biochemistry of liver markers) could be found. Hence, the presence of brown pigment is not assumed to be adverse.

Dose Formulation Analysis
Nominal concentration
The mean of A and B samples showed low values for HD (78 %) and MD (59 %) group in week 1 and a high value for LD (153 %) group in week 1. All other values for weeks 5, 9, and 13 are within a scientifically reasonable range. The individual A or B sample results confirm the mean results of A and B samples.
Since only the first measurement displayed these outlier values regarding nominal concentration and since all other values were within a scientifically acceptable range, it is assumed that this individual measurements at week 1 were outliers and all other concentration are acceptable.
6-h stability
The mean of A and B samples in study week 1 showed increasing amounts for test item after the 6 hours incubation period. In all groups, (LD, MD; HD) a higher amount of test item was determined after 6 hours incubation at room temperature. This was also confirmed by the individual A or B sample results.
No definite explanation for the increasing test item concentrations can be made. Both samples (0h and 6h) are prepared from the same container directly after each other. There is no argument that lithium content should increase in a closed vial. However, since the measured concentration is increasing and not decreasing, this has no influence on the validity of the NOAEL which is mentioned in this study.
Homogeneity investigations
The mean of A and B samples exhibited differences between top/middle/bottom of the test item formulation container from 6-22 %. Except 1 measurement triple (LD) all differences were 20 % or below 20 %. Individual A and B measurements confirm the mean of A and B samples in tendency.
Homogeneity of the test item formulation was within an acceptable range taken into consideration the difficult test sample in water. During the application, test item was continuously vortexed and shaken, especially directly before the application the individual animals.

Li-Determination in Plasma Samples
In male C animals, lithium concentrations between 18.8 µg/L and 128.6 µg/L could be found. In female C animals concentrations between of 3510 µg/L and 6.3 µg/L could be detected. Besides the value of female animal No. 54 (3510 µg/L) all amounts can be attributed to background Lithium values in blood which is attributed e.g. to be 7-24 µg/ml for humans which of course is strongly dependent on respective food which is (besides pharmaceutical intake) the main source of Lithium in the body [9].
In male HD animals, values from 15700 µg/L to 120680 µg/L were found except of one individual animal NO 33 which exhibited 4510 µg/L Lithium. Female HD animals exhibited Serum Lithium values from 10140 µg/L to 128240 µg/L except one individual female No. 82 which exhibited 165 µg/ml.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 100 - < 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
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 material in male and female Wistar rats, with dose levels of 25, 100, and 250 mg/kg body weight day the following conclusions can be made:
At a dose level of 250 mg/kg bw/day food consumption in HD female animals was significantly attenuated. Female HD animals also exhibited increased clinical symptoms like moving the bedding or slight piloerection. Furthermore, weights of adrenals were significantly increased in male HD animals. A significantly decreased thymus weight was recognized for HD female animals, too. Although the decreased food consumption (without significant effect on body weight development) as well as the clinical symptoms cannot be considered as adverse effects alone, together with the significant weight change of adrenals and thymus, these stress symptoms together are conservatively considered to display an adverse effect. Increased iron content, found in serum of HD animals, indicated an increased absorption through intestinal barriers. As a consequence, the transferrin saturation was increased and iron deposition is assumed in liver and kidney, indicated by brown pigment, found in histopathology. Although several pathological consequences are known due to chronic iron deposition in e.g. the liver, within this study no pathological relevance of this was discovered – neither by histopathology nor by clinical biochemistry. Hence, within the present study, the increased iron content in blood serum, the increased transferrin saturation as well as the possible iron deposition in liver and kidney has no toxicological consequence.
At a dose level of 100 mg/kg bw/day no toxicological relevant effect was considered during the period of this study. Hence, the No Observed Adverse Efect Level (NOAEL) is considered to be 100 mg/kg bw/day within this 90 day study.
Executive summary:

The repeated dose toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 408.

During the study the test material 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 female Wistar rats.

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. Organs showing gross alterations were also examined histopathologically. The examinations of these organs were extended to animals of the medium and low dose groups if treatment related changes were observed in high dose group. Additionally, these organs were also examined of the recovery groups.

The following doses were evaluated:

Control:                         0     mg/kg body weight

Low Dose:                   25     mg/kg body weight

Medium Dose:            100    mg/kg body weight

High Dose:                  250    mg/kg body weight

The test material formulation was prepared freshly on each day of administration. The test material 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

One animal of the MD group (No. 75, female) was euthanised due to animal welfare.

The main female animals exhibited more symptoms in HD group compared to the C group or the other treatment group – e.g. 8/10 animals showed moving the bedding, 5/10 animals slight piloerection.

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 as well as recovery period. There were no biologically relevant differences in body temperature between the groups.

During the recovery period a slight tendency to a decreased body weight was recorded for female HD group.

Mean food consumption of female HD animals was significantly attenuated in HD group during the whole treatment period. During the recovery period the mean food consumption was still reduced for HD female animals (p<0.01).

No clear test item related difference could be found for parameters of haematology in treatment groups when compared to C group.

At the end of the treatment period, the amount of iron found in serum of male HD group was significantly (p<0.05) increased. As a consequence, the transferrin saturation was significantly (p<0.05) increased, too. In female animals amount of iron, found in serum, was significantly increased for HD group, too.

Values of AP of female animals at the end of the application period were increased in MD and HD group and the difference of MD vs C group was statistically significant.

No treatment related gross pathological change could be observed during necropsy. 

In males, absolute weights of adrenals were slightly increased in MD (+11 %) and HD (+10 %) animals. This could be confirmed for relative adrenal weights (to body weight but not to brain weight). In particular, an increase of 15 % (MD) and 21 % (HD; p<0.05)) could be mentioned. For Recovery HD group, small increases could be mentioned when compared to recovery C group. In particular, 8 % (absolute), 7 % (relative to brain weight), and 4 % (relative to body weight) were found. For female animals, no change was found for adrenal weights at the end of the treatment period. However, at the end of the recovery period adrenal weights of HD female animals were decreased for 11% (absolute), 9 % (in relation to brain weight), and 6 % (in relation to body weight).

Relative weights (to brain weight) of kidneys of male animals were decreased in HD group (-11 %) when compared to C animals. This was not confirmed for absolute weights or relative weights to body weight. Furthermore, at the end of the recovery period no kidney weight change was mentioned for male HD animals. No weight change of kidneys (absolute and relative) could be recognized for female animals at the end of the treatment period. At the end of the recovery period, a significant decrease in absolute kidney weights was recognized for female HD animals (-10 %; p<0.05). Relative organ weights were slightly decreased (-9% in relation to brain weight and -6% in relation to body weight). A test item relationship cannot be excluded.

In male HD animals, liver weights were significantly increased in relation to body weight (+10 %). This was not confirmed in relation to brain weight or for absolute male liver weights. However, at the end of the recovery period an increase of liver weights could be found in male animals. Absolute weights were increased for 10 % (p<0.05), relative liver weights (to brain weight) were increased for 10 % (p<0.01), and relative liver weights (to body weight) were increased for 6 % (p<0.05). In female animals, relative liver weights (to body weight) were significantly decreased in LD group (-9 %) and MD group (- 9%). No decrease could be found for HD group of relative liver weights (to body weight). This pattern could be confirmed for absolute liver weights and in relation to brain weight (without statistical significance).

In males, absolute weights of thymus were slightly decreased in MD (-10%) and HD (-11%) groups when compared to C group. This was confirmed by relative thymus weights (to brain weight) where a decrease could be mentioned for MD (-17%) as well as HD groups (-18%). Relative thymus weights to body weights were found to be decreased for 10 % (MD) and 6 % (HD). At the end of the recovery period slight decreases of 6 % (absolute weights), 7 % (relative to brain weight), and 10 % (relative to body weight) were observed. In female animals, thymus weights were moderately decreased at the end of the recovery period but not in treatment groups after the treatment period. In recovery C group, -23 % (p<0.05) was observed for absolute thymus weights, -21% for thymus weights in relation to brain weight, and -18% in relation to body weight.

Weights of ovaries of female animals were increased in HD group at the end of the recovery period. In particular, +17% (absolute), +20% (in relation to brain weight), and +23 % (in relation to body weight were mentioned)

Histopathological findings clearly attributable to the test item were seen in the liver and kidney, in females only. In the liver, minimal or mild brown pigment in periportal hepatocytes was observed in females treated at 250 mg/kg/day, against a low background incidence in the control group. In one high dose female, brown pigment was also seen in hepatic macrophages. In females treated at 100 mg/kg/day, a test item relationship could not be excluded for a minimally increased incidence of hepatocellular brown pigment. After the recovery period, liver changes had not resolved. In the kidney, minimal amounts of brown pigment were noted in the corticotubular epithelium in a low number of females treated at 100 or 250 mg/kg/day. This change was no longer seen after the recovery period.

For findings noted in the Peyer’s patch and mesenteric lymph node, at terminal sacrifice only, a test item relationship could not be excluded. In the Peyer’s patch, multifocal brown pigment was noted in a low proportion of treated animals of both sexes, without clear dose relationship, but was also seen in one control male after the recovery period. In animals treated at 250 mg/kg/day, a test item relationship could not be excluded for this change. Likewise, in the mesenteric lymph node, an increased incidence and severity of sinus histiocytosis was noted in the males and females treated at 250 mg/kg/day, when compared to the controls, and was considered to be possibly test item-related.

The mean of nominal concentration in the test item formulation of A and B samples showed low values for HD (78 %) and MD (59 %) group in week 1 and a high value for LD (153 %) group in week 1.

The mean values of the 6-h stability test revealed increasing amounts for test item after the 6 hours incubation period. In all groups, (LD, MD; HD) a higher amount of test item was determined after 6 hours incubation at room temperature.

The mean of A and B samples regarding homogeneity in the test item formulation exhibited differences between top/middle/bottom of the test item formulation container from 6-22 %.

In male C animals, lithium concentrations in plasma were between 18.8 µg/L and 128.6 µg/L. In female C animals concentrations between of 3510 µg/L and 6.3 µg/L could be detected.

In male HD animals, values from 15700 µg/L to 120680 µg/L were found except of one individual animal NO 33 which exhibited 4510 µg/L Lithium. Female HD animals exhibited Serum Lithium values from 10140 µg/L to 128240 µg/L except one individual female No. 82 which exhibited 165 µg/ml.

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