Registration Dossier

Administrative data

Description of key information

Based on an OECD-conform 90 days repated dose oral toxicity study in rats (OECD 408):

NOAEL (male/female rat): 1000 mg/kg bw/d

Key value for chemical safety assessment

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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
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: approx. 7-8 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 155 – 190 g (Mean: 172.38 g, ± 20 % = 137.90 – 206.86 g)
females: 127 – 146 g (Mean: 137.30 g, ± 20 % = 109.84 – 164.76 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities.
Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Only healthy animals were used for this study. Before the first administration all animals used for the study were weighed and assigned to the experimental
groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).


Route of administration:
oral: gavage
Vehicle:
other: 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C).
Details on oral exposure:
The test item, as delivered, was weighed into a tared plastic vial on a suitable precision balance and coated with approx. half the target volume with vehicle.
The vehicle 1 % aqueous hydroxyethyl-cellulose was added to give the appropriate final concentration. The formulation was vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing (Eurofins Munich Study No. 176888), the test item formulations were prepared at least once-weekly
(within stability time frame as given by Eurofins Munich Study No. 176888). The prepared formulation was stored protected from light and at 2-8 °C.
Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.


Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

Administration of Doses
The test item and control formulation were administered at the spezified doses as single application per day to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle
at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 176888).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 176888 (after 30 min without stirring), samples were not collected during the
study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1, 5, 9 and in the last week of treatment (16 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 176889)
and until then stored under appropriate conditions based on available stability data. The B-samples will be retained at < -15 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:
Period of 90 days
Frequency of treatment:
Once a day; 7 days per week
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Additionally, 20 animals (5 males and 5 females per group) were used as recovery animals (control and high dose).
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 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).

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 to identify a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.
Positive control:
not applicable
Observations and examinations performed and frequency:
Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during
the treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days.
The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made once a day, approximately at the same time each day after dosing. The health condition of the 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 cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea,
asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were
made outside the home cage in a standard arena once before the first administration and weekly thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals 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.

Functional Observations
Once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period multiple detailed
behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals..

Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso), large unstained cells (Luc).

Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes..
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT),
alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol),
low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), glucose (Gluc), sodium (Na), potassium (K).
For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and recovery (20 animals) and stored at < -15° C. T3, T4 and TSH serum levels were determined of main study animals (80 animals).

Urinalysis
A urinalysis was performed with samples collected from all animals at necropsy.
The following parameters (specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL),
erythroctes (Ery), leukocytes (Leu)) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Additionally, urine colour / appearance were recorded.

Blood Sampling for potential proof of exposure
For potential proof of exposure after repeated oral administration, blood was sampled from all recovery animals 30 ± 3 minutes after the last treatment (day 90). Approx. 250 µL blood was sampled from the V. Jugularis of slightly anaesthetised rats (Isoflurane) and collected in EDTA- tubes. Samples were placed on ice for potential further processing. Prior to sample analysis an appropriate LC-MS/MS method will be developed for determination of the test item in plasma.








Sacrifice and pathology:
Pathology
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 and xylazin) 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.

Organ Weight
The wet weight of the following organs (liver, uterus, kidneys, thymus, adrenal glands, thyroid/parathyroid glands, testes, spleen, epididymides,
brain, prostate gland, pituitary gland, seminal vesicles and coagulating glands after ligation, heart, ovaries) of all sacrificed animals was
recorded as soon as possible. Paired organs were weighed individually. Organ weights of animal euthanised for animal welfare reasons were
not recorded.

The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenal glands, thyroid/ parathyroid glands, testes, spleen, epididymides,
brain,prostate, seminal vesicles and coagulating glands, pituitary gland, heart, ovaries) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together.
Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.

The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides,
eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular),
lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole,
rectum, salivary glands (sublingual, submandibular, parotis), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow),
stomach, testes, thymus, thyroid gland including parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina) 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.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were also subjected to a gross necropsy and the organs preserved for a histopathological examination.



Histopathology
The afore-listed organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the
roups 1 and 4 sacrificed either at the end of the treatment or recovery period and any animal found dead or euthanised before the planned day of sacrifice.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49,
4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH,
AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance
with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012).
Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

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 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.
Furthermore, statistical comparisons of data acquired during the recovery period were performed with a Dunn’s or Dunnett’s when appropriate.
These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Slight to moderate salivation was noted in all males and females of the HD group, on several days of treatment and in 2/10 males of the MD group. Furthermore, moving the bedding was regularly observed in all males and all females of the HD group and in few males and females of MD group. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore were considered to be unspecific signs of a local reaction to the test item administration rather than a systemic adverse effect.
Alopecia was observed in one male of the MD group and in single or occasional females of the control, LD, MD and HD groups. Crust/wound was observed in few females of the control and dose groups. These findings are mild common background findings and independent from treatment. Dehydration was observed in 5/10 male animals of the LD group on day 7. This was most probably caused by a block of the drinking bottle and independent of treatment. Single or occasional findings, i.e. piloerection, reduced spontaneous activity, abnormal breathing were observed on single or few days in HD male and female animals and are considered to be an incidental findings and not treatment related.
No clinical signs were observed in HD male and female animals during the recovery period. There were no test item related clinical findings during recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no. 62, dosed at 100 mg/kg was found dead in the cage on study day 39. The cause of morbidity was considered to be most likely incidental and not related to the test item administration due to the fact that no histopathological findings that could be attributed to treatment with the test item were observed in this animal. All remaining animals survived until the end of the treatment or recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males and females of all groups, there was an increase of body weight with the progress of the study. Mean body weight change from day 1 to 90 was comparable between the dose groups and control group. Sporadically observed lower / increased body weight gain in male animals of all groups were whitin the range of historical control data and considered not to be of toxicological relevance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean daily food consumption was comparable between all male and female dose groups and controls during the study treatment and recovery period.
On week 1, there was a slightly but statistically significantly lower food consumption in HD males (approx. 5 % below controls) and on week 7, a statistically significant slightly higher food consumption was observed in HD females (approx. 7 % above controls). However, these slight differences were considered incidental and there was no effect of toxicological relevance on food consumption in any of the dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmologic findings were observed in any of the animals of this study.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects of test item were found on all haematological and coagulation parameters of all male and female animals of the dose groups. A statistical significant slight increase in WBC, in male and female animals of MD and HD groups is not considered toxicologically relevant as individual values were within the historical range. No effect of toxicological relevance on haematological and coagulation parameters was found at the end of the recovery period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on clinical biochemistry parameters measured at the end of the treatment and recovery period.
A slight but statistically significant decreased serum creatinine level observed in male and female animals of the HD group, when compared to controls is of no toxicological relevance. A dose response was not observed and a decrease in this level is usually not associated with toxicity. A slight but statistically significant decrease in total Cholesterol and HDL was observed in male animals of the HD group. These changes were not dose related and fully reversible at the end of the recovery period. Thyroid hormon analysis revealed no statistical significant changes beside an increase T4 level only in HD females. However, due to large natural variation in thyroid hormon levels a toxicological significance of this finding is not seen. This view is supported by the absence of changes in the more relevant TSH levels, the restriction of the finding to just one sex and the absence of any histopathological corrolate in the thyroid, especially in high dose females. In addition, all thyroid hormon values in all dose groups are well whithin physiological ranges given in the literature for this strain of rats.
At the end of recovery, a statistically significant slight increase in sodium was observed in male HD group. As levels were within the range of historical control data this is not considered toxicologically relevant. Besides, the mean values for the remaining parameters are within a normal range of variation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item related effects were observed in any of the groups at the end of treatment and recovery period.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
Before initiation of treatment (Week 1), there was statistically significantly lower score of urination in male MD males, a higher count of supported rearings in LD females, non-supported rearings in MD females and higher body temperature in LD females when compared to controls, respectively.
The score for moving in cage and the number of supported rearings in the males of the HD group were statistically significantly increased in week 13 when compared to control group, whereas a statistically significantly lower score for sleeping was observed in these animals. Statistical significantly less supported rearings and lower body temperature was observed in HD group at the end of the recovery period, when compared to the respective controls.
As the effects on various functional observation parameters were slight or inconsistent throughout the dose groups before and at the end of treatment and did not coincide with other parameters of activity or motorcoordination, no toxicological significance can be attributed from these findings.
Immunological findings:
no effects observed
Description (incidence and severity):
No indication of adverse immonological reactions and no observations in immunological relevant organs.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no organ or body weight changes that could be related to treatment with the test item.
A slight but statistically significant higher absolute kidney weight was observed in females of the HD group (10 % above controls), when compared to controls. As no dose response relationship was observed and moreover the changes were not associated with any histopathological findings, this apparent change it is not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross lesions that could be attributed to treatment with the test item. All changes recorded at necropsy are considered to be an incidental finding.
Gross lesion were observed randomly in all the groups at the end of treatment period, like dark lungs, a black left median lobe or dark green spots in the lungs of female animals no. 71 and 77 (MD group) and male animal no. 24 (MD group), liver fused with diaphragm in male animal no. 39 (HD group), a white and hard mass of 1.5 cm at the right kidney of animal no. 34 (HD group), a small right prostate gland in male control animal no. 1, abnormal colored, red spotted thymus in male Control animal no. 4 and male animal no. 24 of the MD group and small, complete thymus of MD group male animal no. 24 and abnormally colored and spotted mesenteric lymph node in 2/10 HD females. Uterus with dilatation on both sides was seen in 2/10 control animals, 2/10 animals of the LD group, 3/10 animals of the MD group and 5/10 animals of the HD group. All these are not considered to be toxicologically relevant.
Gross lesions were also observed at the end of recovery period in HD groups, i.e. a small prostate gland, (1/5 animals), cervix dilatation in 1/5 control animals, small right adrenal gland with abnormal color, in 1/5 HD males (no. 99) and abnormal color spotted mesenteric lymph node in 2/5 control and 2/5 males of the HD group. Based on histopathological evaluation the gross lesions were considered to be incidental and not related to treatment with the test item.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No effects observed in any parameter of the functional observation battery.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histopathological findings that could be attributed to treatment with the test item. All recorded findings were considered incidental or
were within the range of background alterations that may be recorded in Wistar rats of this age.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic findings observed during histopathology.
Details on results:
Mortality
Female no. 62, dosed at 100 mg/kg was found dead in the cage on study day 39. A contribution of the macroscopically observed diaphragmatic hernia of the liver cannot be excluded.
However, the cause of morbidity was considered to be most likely incidental and not related to the test item administration. All remaining animals survived until the end of the treatment or recovery period.

Clinical Observations
There were no clinical signs of systemic toxicity in this study.
Slight to moderate salivation was noted in all males and females of the HD group, on several days of treatment and in 2/10 males of the MD group. Furthermore, moving the bedding
was regularly observed in all males and all females of the HD group and in few males and females of MD group. The clinical signs salivation and moving the bedding were observed
in close timely relation to the dose administration and therefore were considered to be signs of a local reaction to the test item rather than a systemic adverse effect.
Alopecia was observed in one male of the MD group and in single or occasional females of the control, LD, MD and HD groups. Crust/wound was observed in few females of the
control and dose groups. These findings are mild common background findings and independent from treatment. Dehydration was observed in 5/10 male animals of the LD group on day 7.
This was most probably caused by a block of the drinking bottle and independent of treatment. Single or occasional findings, i.e. piloerection, reduced spontaneous activity,
abnormal breathing were observed on single or few days in HD male and female animals and are considered to be an incidental findings and not treatment related.
No clinical signs were observed in HD male and female animals during the recovery period.
During the weekly detailed clinical observation, no considerable differences between the groups were found except statistically significant softer faecal consistency
and a lower score for sleeping in the cage in week 1 and a higher score for moving in cages in week 1, higher score of sleeping in the cage and a lower score for moving in cages in week 3.
The statistically significant differences in detailed clinical examination parameters were not considered to be treatment related, as they were observed occasionally without dose dependency.
There were no test item related clinical findings during recovery period.

Functional Observation Battery
No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment.
Before initiation of treatment (Week 1), there was statistically significantly lower score of urination in male MD males, a higher count of supported rearings in LD females, non-supported rearings in
MD females and higher body temperature in LD females when compared to controls, respectively.
The score for moving in cage and the number of supported rearings in the males of the HD group were statistically significantly increased in week 13 when compared to control group,
whereas a statistically significantly lower score for sleeping was observed in these animals. Statistical significantly less supported rearings and lower body temperature was observed in
HD group at the end of the recovery period, when compared to the respective controls.
As the effects on various functional observation parameters were slight or inconsistent throughout the dose groups before and at the end of treatment and did not coincide with other
parameters of activity or motorcoordination, no toxicological significance can be attributed from these findings.

Ophthalmoscopic Findings
No ophthalmologic findings were observed in any of the animals of this study.

Body Weight Development
In males and females of all groups, there was an increase of body weight with the progress of the study. Mean body weight change from day 1 to 90 was comparable between the dose groups
and control group. In the HD males, moderate and statistically significantly lower body weight gain was seen in week 11 of treatment when compared to control group (46 % below control)
and in males of LD and MD group moderate and statistically significantly increased body weight gain in week 3 (45% and 24% above control, respectively). In females, no statistically or biologically
significant differences were noted for body weight gain between the dose groups and the control group.
During the recovery period, between day 90 to 118 body weight change was statistically significantly higher in male but not female animals of the HD group, when compared to controls
(88.57% in week 14, 88.6% in week 16 and, 71.42% in week 13). As the body weights were within the range historical control data, this is not assumed to be toxicologically relevant.

Food Consumption
Mean daily food consumption was comparable between all male and female dose groups and controls during the study treatment and recovery period.
On week 1, there was a slightly but statistically significantly lower food consumption in HD males (approx. 5 % below controls) and on week 7, a statistically significant slightly higher
food consumption was observed in HD females (approx. 7 % above controls). However, these slight differences were considered incidental and there was no effect of toxicological relevance
on food consumption in any of the dose groups.

Haematology and Blood Coagulation
No adverse test item related effects were found for all haematological and coagulation parameters for all male and female dose groups at the end of the treatment and recovery period.
A statistically significantly slight increase in WBC in male and females of MD and HD groups is not considered to be adverse as individual values were within the historical control data range.
However, a relation to the test item cannot be excluded. A statistically significant slightly higher Hb and MCHC in females of HD group was observed at the end of the treatment period.
As values were within the range of historical control data this is not considered toxicologically relevant. A tendency towards lower Eos in males and higher Eos in females of the dose
groups is not considered to be toxicologically relevant.
At the end of the recovery period, a slight statistically non-significant increase in WBC in males (33.60 % above control) and a slight but statistical significant slight decrease in WBC in females
of the HD group (36.66 % below control) were observed. As values were within the range of historical control data and due to the inconsistency, these differences are not considered toxicologically relevant.
A statistically significant slightly lower MCH in males of HD group was observed at the end of the recovery period. As values were within the range of historical control data this is not
considered toxicologically relevant.

Clinical Biochemistry
There was no test item related effect on clinical biochemistry parameters measured at the end of the treatment and recovery period.
A slight but statistically significant decreased serum creatinine level observed in male and female animals of the HD group, when compared to controls is of no toxicological relevance. A decrease in this level is usually not associated with toxicity. A slight but statistically significant decrease in total Cholesterol and HDL was observed in male animals of the HD group. A relation to the test item cannot be excluded. Thyroid hormon analysis revealed no statistical significant changes beside an increase T4 level only in HD females. However, due to large natural variation in thyroid hormon levels a toxicological significance of this finding is not seen. This view is supported by the absence of changes in the more relevant TSH levels, the restriction of the finding to just one sex and the absence of any histopathological corrolate in the thyroid, especially in high dose females. In addition, all thyroid hormon values in all dose groups are well whithin physiological ranges given in the literature for this strain of rats.

At the end of recovery, a statistically significant slight increase in sodium was observed in male HD group.
As levels were within the range of historical control data this is not considered toxicologically relevant.
Besides, the mean values for the remaining parameters are within a normal range of variation.


Urinalysis
There were no test item related effects were observed in any of the groups at the end of treatment and recovery period.

Pathology
There were no gross lesions that could be attributed to treatment with the test item. All changes recorded at necropsy are considered to be an incidental finding.
Gross lesion were observed randomly in all the groups at the end of treatment period, like dark lungs, a black left median lobe or dark green spots in the lungs of female animals no. 71 and 77 (MD group) and male animal no. 24 (MD group), liver fused with diaphragm in male animal no. 39 (HD group), a white and hard mass of 1.5 cm at the right kidney of animal no. 34 (HD group), a small right prostate gland in male control animal no. 1, abnormal colored, red spotted thymus in male Control animal no. 4 and male animal no. 24 of the MD group and small, complete thymus of MD group male animal no. 24 and abnormally colored and spotted mesenteric lymph node in 2/10 HD females. Uterus with dilatation on both sides was seen in 2/10 control animals, 2/10 animals of the LD group, 3/10 animals of the MD group and 5/10 animals of the HD group. All these are not considered to be toxicologically relevant. Gross lesions were also observed at the end of recovery period in HD groups, i.e. a small prostate gland, (1/5 animals), cervix dilatation in 1/5 control animals,
small right adrenal gland with abnormal color, in 1/5 HD males (no. 99) and abnormal color spotted mesenteric lymph node in 2/5 control and 2/5 males of the HD group.
Based on histopathological evaluation the gross lesions were considered to be incidental and not related to treatment with the test item.


Organ Weight
There were no organ or body weight changes that could be related to treatment with the test item.
A slight but statistically significant higher absolute kidney weight was observed in females of the HD group (10 % above controls), when compared to controls.
As this was not associated with any histopathological findings, it is not considered to be toxicologically relevant.
Besides, there were no statistically significant differences in weights of any of the other organs between control and dose groups.
A tendency towards lower absolute thyroid/parathyroid weight of male animals of the HD group (25 % below controls) and higher thymus weight in
female animals of the HD group (17 % above controls) were not associated with histopathological findings and are not considered to be toxicologically relevant.


Histopathology
There were no histopathological findings that could be attributed to treatment with the test item. All recorded findings were considered incidental or
were within the range of background alterations that may be recorded in Wistar rats of this age.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 9 and 13 of the study.
The mean recoveries observed for the LD dose group were between 101.6 % and 121.0 % of the nominal value, between 89.7% and 107.8% of the nominal value for the MD dose group and between 86.7 % and 109.2 % of the nominal value for HD dose group. In week 13, the mean recovery observed for the LD was slightly higher (121 %) than the nominal value. However, the overall mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 109.1 %, 97.4 %, and 97.6 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.




Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related signs of toxicity were obseved
Key result
Critical effects observed:
no

Mortality

 

Sex:Male- Phase:In-life

 

 

 

 

 

 

 

 

C/M

LD/M

MD/M

HD/M

 

Animals examined

N

15

10

10

15

day1->118

Alive

N

15

10

10

15

 

Alive

%

100.0

100.0

100.0

100.0


Mortality

 

 

Sex:Female-Phase:In-life

 

 

 

 

 

 

 

 

 

C/M

LD/M

MD/M

HD/M

 

Animals examined

N

15

10

10

15

day1->118

Alive

N

15

9

10

15

 

Alive

%

100.0

90.00

100.0

100.0

Clinical Biochemistry

Sex:Male- Phase:In-life

 

C / M

LD / M

MD / M

HD / M

ALAT

Mean

36.21

36.82

34.78

36.24

[U/L]

S.d.

5.935

5.677

4.268

6.667

day 91

N

10

10

10

9

Gluc

Mean

11.243

12.370

13.107

12.109

[mmol/L]

S.d.

2.6142

2.1722

3.0591

1.8560

day 91

N

10

10

10

9

Crea

Mean

21.3

19.5

22.4

17.7

[µmol/L]

S.d.

2.71

3.84

7.65

1.66

day 91

N

10

10

10

9

ASAT

Mean

116.15

103.21

99.23

92.31

[U/L]

S.d.

29.435

14.777

19.046

17.059

day 91

N

10

10

10

9

AP

Mean

122.795

125.367

113.942

118.826

[U/L]

S.d.

24.8284

36.0696

35.6057

44.7871

day 91

N

10

10

10

9

Na

Mean

142.3

141.2

140.7

142.0

[mmol/L]

S.d.

2.75

3.08

2.75

1.66

day 91

N

10

10

10

9

K

Mean

4.949

4.742

4.798

5.006

[mmol/L]

S.d.

0.6791

0.7578

0.8119

0.6875

day 91

N

10

10

10

9

TP

Mean

59.84

61.12

59.23

57.98

[g/L]

S.d.

2.991

3.556

3.165

1.731

day 91

N

10

10

10

9

ALB

Mean

32.702

33.676

32.144

32.378

[g/L]

S.d.

1.8167

1.8430

1.4392

0.9289

day 91

N

10

10

10

9

 

 

C / M

LD / M

MD / M

HD / M

TBIL

Mean

2.01

2.08

1.91

2.07

[µmol/L]

S.d.

0.285

0.466

0.335

0.250

day 91

N

10

10

10

9

CHOL

Mean

2.014

1.957

2.020

1.661

[mmol/L]

S.d.

0.2878

0.1357

0.1890

0.2318

day 91

N

10

10

10

9

TBA

Mean

17.878

22.622

22.256

18.074

[µmol/L]

S.d.

5.9091

10.0550

7.2659

6.8755

day 91

N

9

10

10

9

TG

Mean

0.97

1.09

1.26

1.46

[mmol/L]

S.d.

0.352

0.405

0.371

0.431

day 91

N

8

10

9

9

Urea

Mean

7.261

7.216

7.556

6.232

[mmol/L]

S.d.

1.1827

0.7894

1.0914

0.7559

day 91

N

10

10

10

9

HDL

Mean

1.284

1.296

1.311

1.076

[mmol/L]

S.d.

0.1361

0.1059

0.1073

0.1680

day 91

N

10

10

10

9

LDL (calculated)

Mean

0.528

0.443

0.458

0.294

[mmol/L]

S.d.

0.1402

0.0696

0.1156

0.1328

day 91

N

8

10

9

9

ALAT

Mean

34.62

 

 

26.95

[U/L]

S.d.

5.798

 

 

4.268

day 119

N

5

0

0

4

Gluc

Mean

15.076

 

 

12.480

[mmol/L]

S.d.

2.7719

 

 

2.1391

day 119

N

5

0

0

4

 

C / M

LD / M

MD / M

HD / M

Crea

Mean

28.4

 

 

27.0

[µmol/L]

S.d.

9.07

 

 

3.46

day 119

N

5

0

0

4

ASAT

Mean

94.34

 

 

96.32

[U/L]

S.d.

20.240

 

 

16.872

day 119

N

5

0

0

4

AP

Mean

93.424

 

 

81.502

[U/L]

S.d.

23.9905

 

 

34.2293

day 119

N

5

0

0

4

Na

Mean

140.6

 

 

143.5

[mmol/L]

S.d.

1.14

 

 

1.29

day 119

N

5

0

0

4

K

Mean

4.590

 

 

4.110

[mmol/L]

S.d.

0.4584

 

 

0.4334

day 119

N

5

0

0

4

TP

Mean

53.36

 

 

55.52

[g/L]

S.d.

3.304

 

 

1.936

day 119

N

5

0

0

4

ALB

Mean

31.116

 

 

32.168

[g/L]

S.d.

2.0129

 

 

1.1706

day 119

N

5

0

0

4

TBIL

Mean

1.84

 

 

1.82

[µmol/L]

S.d.

0.251

 

 

0.206

day 119

N

5

0

0

4

CHOL

Mean

1.666

 

 

1.852

[mmol/L]

S.d.

0.1941

 

 

0.1179

day 119

N

5

0

0

4

 

 

C / M

LD / M

MD / M

HD / M

TBA

Mean

21.266

 

 

19.305

[µmol/L]

S.d.

7.6762

 

 

7.8503

day 119

N

5

0

0

4

TG

Mean

0.90

 

 

1.00

[mmol/L]

S.d.

0.254

 

 

0.160

day 119

N

5

0

0

4

Urea

Mean

8.392

 

 

6.300

[mmol/L]

S.d.

1.7579

 

 

1.0450

day 119

N

5

0

0

4

HDL

Mean

1.110

 

 

1.218

[mmol/L]

S.d.

0.0970

 

 

0.0981

day 119

N

5

0

0

4

LDL (calculated)

Mean

0.376

 

 

0.436

[mmol/L]

S.d.

0.1349

 

 

0.0322

day 119

N

5

0

0

4

Sex:Female- Phase:In-life

 

C / F

LD / F

MD / F

HD / F

ALAT

Mean

32.96

31.92

32.43

30.57

[U/L]

S.d.

6.598

5.738

4.585

9.530

day 91

N

10

9

10

10

Gluc

Mean

10.500

9.811

10.836

9.920

[mmol/L]

S.d.

1.9542

1.6568

1.7542

2.2668

day 91

N

10

9

10

10

Crea

Mean

26.0

22.3

22.9

21.3

[mmol/L]

S.d.

3.59

2.83

3.75

3.09

day 91

N

10

9

10

10

ASAT

Mean

80.37

80.93

75.79

73.90

[U/L]

S.d.

5.358

13.419

10.277

14.983

day 91

N

10

9

10

10

AP

Mean

70.535

79.556

69.461

60.147

[U/L]

S.d.

37.3140

37.5844

20.6289

16.6869

day 91

N

10

9

10

10

Na

Mean

143.7

143.3

142.7

143.3

[mmol/L]

S.d.

1.77

2.50

1.77

2.54

day 91

N

10

9

10

10

K

Mean

3.664

3.690

3.873

3.627

[mmol/L]

S.d.

0.2393

0.2533

0.3836

0.1601

day 91

N

10

9

10

10

TP

Mean

59.77

61.06

60.39

61.06

[g/L]

S.d.

3.540

2.905

3.213

3.737

day 91

N

10

9

10

10

ALB

Mean

33.824

34.467

34.195

34.611

[g/L]

S.d.

2.1958

1.4768

1.8785

1.9025

day 91

N

10

9

10

10

 

 

C / F

LD / F

MD / F

HD / F

TBIL

Mean

2.30

2.36

2.70

2.77

[µmol/L]

S.d.

0.598

0.340

0.478

0.521

day 91

N

10

9

10

10

CHOL

Mean

1.362

1.344

1.311

1.276

[mmol/L]

S.d.

0.3601

0.2532

0.3015

0.3420

day 91

N

10

9

10

10

TBA

Mean

14.322

13.810

14.748

15.200

[µmol/L]

S.d.

7.7652

4.3278

6.1424

13.3457

day 91

N

10

9

10

10

TG

Mean

0.82

0.91

0.91

0.94

[mmol/L]

S.d.

0.329

0.322

0.349

0.380

day 91

N

10

8

10

10

Urea

Mean

6.909

6.532

6.004

6.652

[mmol/L]

S.d.

0.7241

0.6999

0.5171

1.0934

day 91

N

10

9

10

10

HDL

Mean

0.948

0.929

0.885

0.828

[mmol/L]

S.d.

0.2581

0.2037

0.2218

0.3040

day 91

N

10

9

10

10

LDL (calculated)

Mean

0.250

0.233

0.244

0.261

[mmol/L]

S.d.

0.1115

0.1055

0.1220

0.0964

day 91

N

10

8

10

10

ALAT

Mean

30.98

 

 

22.54

[U/L]

S.d.

8.708

 

 

3.868

day 119

N

5

0

0

5

Gluc

Mean

9.988

 

 

8.180

[mmol/L]

S.d.

1.7758

 

 

0.9301

day 119

N

5

0

0

5

 

C / F

LD / F

MD / F

HD / F

Crea

Mean

36.6

 

 

25.6

[mmol/L]

S.d.

23.41

 

 

1.14

day 119

N

5

0

0

5

ASAT

Mean

81.82

 

 

68.90

[U/L]

S.d.

19.355

 

 

15.175

day 119

N

5

0

0

5

AP

Mean

51.558

 

 

35.658

[U/L]

S.d.

10.2563

 

 

13.7959

day 119

N

5

0

0

5

Na

Mean

139.8

 

 

141.8

[mmol/L]

S.d.

3.49

 

 

1.48

day 119

N

5

0

0

5

K

Mean

3.822

 

 

3.756

[mmol/L]

S.d.

0.4126

 

 

0.2085

day 119

N

5

0

0

5

TP

Mean

59.14

 

 

61.20

[g/L]

S.d.

2.827

 

 

3.798

day 119

N

5

0

0

5

ALB

Mean

34.914

 

 

36.208

[g/L]

S.d.

1.6809

 

 

2.6896

day 119

N

5

0

0

5

TBIL

Mean

2.84

 

 

2.64

[µmol/L]

S.d.

0.699

 

 

0.706

day 119

N

5

0

0

5

CHOL

Mean

1.118

 

 

1.302

[mmol/L]

S.d.

0.2750

 

 

0.0887

day 119

N

5

0

0

5

 

 

C / F

LD / F

MD / F

HD / F

TBA

Mean

22.076

 

 

17.520

[µmol/L]

S.d.

9.8022

 

 

7.3873

day 119

N

5

0

0

5

TG

Mean

0.55

 

 

0.60

[mmol/L]

S.d.

0.182

 

 

0.142

day 119

N

5

0

0

5

Urea

Mean

7.480

 

 

5.984

[mmol/L]

S.d.

2.6890

 

 

0.7884

day 119

N

5

0

0

5

HDL

Mean

0.756

 

 

0.892

[mmol/L]

S.d.

0.2341

 

 

0.0826

day 119

N

5

0

0

5

LDL (calculated)

Mean

0.252

 

 

0.290

[mmol/L]

S.d.

0.0611

 

 

0.0229

day 119

N

5

0

0

5

 

Summary Body weight (g)- male - Phase: in -life

 

C/M

LD/M

MD/M

HD/M

day1

Mean

220.47a

219.10

224.70

222.93

 

S.d.

10.38

12.27

9.67

13.40

N                                                                                         15                      10                        10                       15

 

DeviationVsControl[%]

 

-0.62

1.92

1.12

day8

Mean

255.60k

231.80

257.90

255.87

 

S.d.

13.41

24.53

10.80

15.70

N                                                         15                      10                        10                 15

 

DeviationVsControl[%]

 

-9.31

0.90

0.10

day15

Mean

284.73a

271.20

286.70

285.13

 

S.d.

18.05

16.81

12.63

16.58

N                                                                                         15                      10                        10                       15

 

DeviationVsControl[%]

 

-4.75

0.69

0.14

day22

Mean

307.13a

303.60

314.50

309.40

 

S.d.

21.46

17.03

14.44

17.88

N                                                        15                      10                        10                  15

 

DeviationVsControl[%]

 

-1.15

2.40

0.74

day29

Mean

321.93a

321.80

328.20

325.07

 

S.d.

24.02

17.83

17.69

19.59

N                                                     15                      10                        10                       15

 

DeviationVsControl[%]

 

-0.04

1.95

0.97

day36

Mean

335.13a

337.00

345.30

341.33

 

S.d.

25.39

19.74

18.52

18.80

N                                15                      10                        10                       15

 

DeviationVsControl[%]

 

0.56

3.03

1.85

day43

Mean

346.47a

347.70

357.30

352.93

 

S.d.

27.14

21.03

19.26

21.40

N                                                          15                      10                        10                  15

 

DeviationVsControl[%]

 

0.36

3.13

1.87

day50

Mean

356.00a

359.90

364.10

362.93

 

S.d.

27.38

23.96

19.81

24.52

N                                                           15                      10                        10             15

 

DeviationVsControl[%]

 

1.10

2.28

1.95

day57

Mean

364.33a

367.00

375.00

369.40

 

S.d.

29.41

23.63

20.78

25.56

N                                                                15                      10                    10        15

 

DeviationVsControl[%]

 

0.73

2.93

1.39

day64

Mean

371.27a

377.00

383.30

377.07

 

S.d.

31.47

24.24

24.98

26.65

N                                                                    15                 10              10                  15

 

DeviationVsControl[%]

 

1.54

3.24

1.56

day71

Mean

376.67a

382.40

391.00

384.87

 

S.d.

30.98

24.13

28.49

26.99

 

N

15

10

10

15

 

DeviationVsControl[%]

 

1.52

3.81

2.18

 

C/M

LD/M

MD/M

HD/M

day78

Mean

387.33a

393.30

399.10

390.67

 

S.d.

31.68

24.18

27.79

27.86

N                                  15           10            10             15

 

DeviationVsControl[%]

 

1.54

3.04

0.86

day85

Mean

390.33a

397.30

403.80

394.93

 

S.d.

32.16

24.53

29.17

31.04

N                           15              10                        10             15

 

DeviationVsControl[%]

 

1.78

3.45

1.18

day90

Mean

397.47a

410.80

410.00

400.27

 

S.d.

35.08

25.53

30.76

33.19

 

N

15

10

10

15

 

DeviationVsControl[%]

 

3.35

3.15

0.70

 

C/F

LD/F

MD/F

HD/F

day1

Mean

161.80k

163.90

166.20

162.38

 

S.d.

7.04

8.84

11.04

6.15

 

N

15

10

10

16

 

DeviationVsControl[%]

 

1.30

2.72

0.36

day8

Mean

176.93a

176.60

176.90

177.07

 

S.d.

8.58

9.97

11.35

9.66

 

N

15

10

10

15

 

DeviationVsControl[%]

 

-0.19

-0.02

0.08

day15

Mean

189.00a

189.20

191.10

189.40

 

S.d.

10.60

11.04

13.46

10.20

N                                                15                  10                      10              15

 

DeviationVsControl[%]

 

0.11

1.11

0.21

day22

Mean

197.60a

199.20

200.10

196.67

 

S.d.

9.49

8.82

13.83

12.83

 

N

15

10

10

15

 

DeviationVsControl[%]

 

0.81

1.27

-0.47

day29

Mean

206.53a

209.80

209.20

209.00

 

S.d.

11.09

9.67

15.17

9.96

 

N

15

10

10

15

 

DeviationVsControl[%]

 

1.58

1.29

1.19

day36         Mean                                               213.93a            217.60               214.60              214.67

 

S.d.

9.48

11.17

13.47

11.78

 

N

15

10

10

15

 

DeviationVsControl[%]

 

1.71

0.31

0.34

day43

Mean

215.27k

220.22

219.80

217.13

 

S.d.

10.44

13.24

16.22

11.97

N                                                    15                         9                      10                     15

 

DeviationVsControl[%]

 

2.30

2.11

0.87

day50

Mean

218.67k

222.44

220.90

220.40

 

S.d.

11.90

9.76

14.12

10.36

N                                                                  15                    9                 10                   15

 

DeviationVsControl[%]

 

1.73

1.02

0.79

day57

Mean

221.47k

225.78

225.10

224.00

 

S.d.

10.97

10.95

14.72

9.11

N                                                           15                        9                      10             15

 

DeviationVsControl[%]

 

1.95

1.64

1.14

day64

Mean

224.80a

231.78

228.50

228.53

 

S.d.

11.83

13.92

16.08

14.15

N                                                    15                 9                      10                15

 

DeviationVsControl[%]

 

3.10

1.65

1.66

day71

Mean

229.80k

233.11

233.80

232.00

 

S.d.

13.11

10.73

14.11

13.93

 

N

15

9

10

15

 

DeviationVsControl[%]

 

1.44

1.74

0.96

 

C/F

LD/F

MD/F

HD/F

day78

Mean

230.27a

235.00

235.60

234.40

 

S.d.

13.77

10.61

16.75

13.39

N                                                       15                        9                      10                       15

 

DeviationVsControl[%]

 

2.06

2.32

1.80

day85

Mean

230.73a

238.00

238.70

238.00

 

S.d.

12.87

12.50

17.27

12.42

N                                                       15                        9                      10                       15

 

DeviationVsControl[%]

 

3.15

3.45

3.15

day90

Mean

232.87a

238.44

237.30

236.93

 

S.d.

12.78

12.10

15.85

15.61

 

N

15

9

10

15

 

DeviationVsControl[%]

 

2.40

1.90

1.75

Summary of Hematology

Male- Phase: in-life

 

C/M

LD/M

MD/M

HD/M

WBC

Mean

4.1450ad

4.9411

5.7090*

6.3489**

[1E9/L]

S.d.

1.0948

0.9088

1.0546

2.0651

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

19.2065

37.7322

53.1698

RBC               Mean                                         9.6040k              9.6878                9.9930                9.9444

[1E12/L]

S.d.

0.4133

0.2589

0.3748

0.3792

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

0.8723

4.0504

3.5448

HGB               Mean                                           16.350k           16.556                16.800          16.989

[g/dL]

S.d.

0.688

0.579

0.855

0.280

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

1.257

2.752

3.908

HCT                Mean                                            52.610k           52.711          53.620                54.100

[%]

S.d.

2.416

1.437

2.711

1.672

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

0.192

1.920

2.832

MCV               Mean                                             54.770a              54.422             53.650          54.433

[fL]

S.d.

0.910

1.861

1.377

1.713

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

-0.635

-2.045

-0.615

MCH               Mean                                        17.000a               17.100                16.820            17.100

[pg]

S.d.

0.170

0.696

0.421

0.574

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

0.588

-1.059

0.588

MCHC            Mean                                           31.110a              31.422                31.340            31.411

[g/dL]

S.d.

0.615

0.800

0.723

0.717

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

1.004

0.739

0.968

RET%            Mean                                                1.6520k          1.6744       1.7510                1.6256

[%]

S.d.

0.4626

0.2351

0.2942

0.2815

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

1.3586

5.9927

-1.6008

 

C/M

LD/M

MD/M

HD/M

PLT

Mean

635.80a

622.78

659.70

624.22

[1E9/L]

S.d.

69.24

126.33

81.55

68.87

day91             N                                                                              10                           9                         10                            9

 

DeviationVsControl[%]

 

-2.05

3.76

-1.82

EOS%

Mean

0.6300k

0.3333

0.3600

0.2778

[%]

S.d.

0.6273

0.2449

0.2989

0.1856

day91             N                                                                              10                           9                         10                            9

 

DeviationVsControl[%]

 

-47.0899

-42.8571

-55.9083

LYM%

Mean

77.870a

76.667

79.230

78.322

[%]

S.d.

5.447

8.829

3.746

4.614

day91             N                                                       10                   9                 10                         9

 

DeviationVsControl[%]

 

-1.545

1.747

0.581

NEUT%

Mean

18.990a

20.478

16.950

19.078

[%]

S.d.

5.007

8.037

2.668

4.135

day91

N

10

9

8

9

 

DeviationVsControl[%]

 

7.835

-10.742

0.462

MONO%

Mean

2.220a

2.189

2.012

1.944

[%]

S.d.

0.603

0.779

0.617

0.498

day91

N

10

9

8

9

 

DeviationVsControl[%]

 

-1.401

-9.347

-12.412

BASO%

Mean

0.1100k

0.1000

0.1000

0.1222

[%]

S.d.

0.0876

0.0866

0.0667

0.0667

day91

N

10

9

10

9

 

DeviationVsControl[%]

 

-9.0909

-9.0909

11.1111

LUC%             Mean                                            0.1700k          0.2111          0.2250                 0.2333

[%]

S.d.

0.1059

0.1269

0.1035

0.0866

day91

N

10

9

8

9

 

DeviationVsControl[%]

 

24.1830

32.3529

37.2549

Sex: female

Phase: Life-in

 

C/F

LD/F

MD/F

HD/F

WBC

Mean

2.7910ad

2.8133

3.8510*

3.7720*

[1E9/L]

S.d.

0.3375

0.7408

0.9480

0.9162

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

0.8002

37.9792

35.1487

RBC                Mean                                                8.3350a          8.5889        8.2960                8.6410

[1E12/L]

S.d.

0.3947

0.4125

0.4556

0.4314

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

3.0461

-0.4679

3.6713

HGB                Mean                                                14.840ad           15.411         15.060          15.530*

[g/dL]

S.d.

0.544

0.542

0.597

0.587

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

3.848

1.482

4.650

HCT                Mean                                            47.620a             48.867                 47.560            48.490

[%]

S.d.

1.997

2.037

2.166

2.155

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

2.618

-0.126

1.827

MCV               Mean                                               57.160k             56.933           57.370        56.130

[fL]

S.d.

1.512

1.546

1.569

1.431

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

-0.397

0.367

-1.802

MCH               Mean                                               17.830a             17.956           18.170             18.000

[pg]

S.d.

0.558

0.546

0.611

0.618

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

0.704

1.907

0.953

MCHC            Mean                                        31.170ad           31.556                 31.670         32.070**

[g/dL]

S.d.

0.435

0.553

0.501

0.653

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

1.237

1.604

2.887

RET%             Mean                                         1.9380a             1.8556                 1.8470                1.8300

[%]

S.d.

0.3466

0.2912

0.4640

0.2517

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

-4.2541

-4.6956

-5.5728

Sex:Fe

male-Phase:In-life

 

 

 

C/F

LD/F

MD/F

HD/F

PLT[1E9/L]

Mean

S.d.

644.80k

60.97

548.33

185.07

639.00

100.47

625.40

118.12

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

-14.96

-0.90

-3.01

EOS%[%]

Mean

S.d.

0.2200k

0.1476

0.2667

0.2062

0.3200

0.2486

0.4600

0.5929

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

21.2121

45.4545

109.0909

LYM%[%]

Mean

S.d.

83.180k

4.415

81.478

6.798

81.990

6.503

81.990

7.776

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

-2.046

-1.431

-1.431

NEUT[%]

%           Mean

S.d.

14.630a

3.984

15.889

6.402

15.520

6.026

15.030

7.338

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

8.605

6.083

2.734

MONO%

Mean

1.760a

2.011

1.770

2.200

[%]

S.d.

0.508

0.499

0.365

0.455

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

14.268

0.568

25.000

BASO%

Mean

0.0700k

0.1000

0.0500

0.0600

[%]

S.d.

0.0675

0.1118

0.0850

0.0516

day91

N

10

9

10

10

 

DeviationVsControl[%]

 

42.8571

-28.5714

-14.2857

LUC%             Mean                                             0.1500a             0.2111        0.3000                   0.2800

[%]

 

S.d.

0.1080

0.1167

0.1414

0.1687

day91

 

N

10

9

10

10

 

 

DeviationVsControl[%]

 

40.7407

100.0000

86.6667

Histopathological data summary attached as pdf

Conclusions:
On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Propyl 4-hydroxybenzoate in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made: The no observed adverse effect level (NOAEL) of Propyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day.

Executive summary:

The objective of this study was to assess the possible health hazards which could arise from repeated exposure of Propyl 4-hydroxybenzoate via oral administration to rats over a period of 90 consecutive 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 1 % aqueous hydroxyethyl-cellulose, the vehicle used in this study. The 4 groups comprised of 10 male and 10 female Wistar rats. Control, low, mid and high dose group rats received the dose at 0, 100, 300 and 1000 mg/kg/day respectively, as repeated dose at the dose volume of 5 mL/kg. The test item formulation was prepared at least every 4 days. The test item was suspended in 1 % aqueoushydroxyethyl-cellulose 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.

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.

functional observation battery and ophthalmoscopy examination did not reveal any test item related effects in any of the treatment and recovery groups.

In males and females, there was no test item related effect on body weight during both treatment and recovery period.

There was no effect of toxicological relevance on food consumption in any of the dose groups during both treatment and recovery period.

No toxicologically relevant effects of test item were found on all haematological and coagulation parameters of all male and female animals of the dose groups. A statistical significant slight increase in WBC, in male and female animals of MD and HD groups is not considered toxicologically relevant as individual values were within the historical range. No effect of toxicological relevance on haematological and coagulation parameters was found at the end of the recovery period. There was no adverse effect on clinical biochemistry parameters measured at the end of the treatment and recovery period. Changes in some clinical biochemistry parameter were in most cases not dose related and not considered to be of toxicological relevance. There was no adverse effect on urinary parameters measured at the end of the treatment and recovery period. Based on histopathological evaluation, none of the occasional gross lesions observed at necropsy were considered to be related to treatment with test iteml. No statistically significant difference in organ weight was found in male and female animals at any of the dose levels tested when compared to control. At histopathological evaluation, there were no abnormalities that could be attributed to treatment with the test items.

Conclusion

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Propyl 4-hydroxybenzoate in male and femaleWistarrats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

The no observed adverse effect level (NOAEL) of Propyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

Propyl paraben 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 1 % aqueoushydroxyethyl-cellulose, the vehicle used in this study. The 4 groups comprised of 10 male and 10 female Wistarrats.Control, low, mid and high dose group rats received the dose at 0, 100, 300 and 1000 mg/kg/day respectively, as repeated dose at the dose volume of 5 mL/kg. 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.

There were no test item related clinical signs of systemic toxicity during the treatment and recovery period in any of the animals. In addition, the detailed clinical examination, functional observation battery and ophthalmoscopy examination did not reveal any test item related effects in any of the treatment and recovery groups.

In males and females, there was no test item related effect on body weight during both treatment and recovery period.There was no effect of toxicological relevance on food consumption in any of the dose groups during both treatment and recovery period.

No toxicologically relevant effects of test item were found on all haematological and coagulation parameters of all male and female animals of the dose groups.

A statistical significant slight increase in WBC, in male and female animals of MD and HD groups is not considered toxicologically relevant as individual values were within the historical range. No effect of toxicological relevance on haematological and coagulation parameters was found at the end of the recovery period.

There was no adverse effect on clinical biochemistry parameters measured at the end of the treatment and recovery period. Changes in some clinical biochemistry parameter were in most cases not dose related and not considered to be of toxicological relevance. There was no adverse effect on urinary parameters measured at the end of the treatment and recovery period. Based on histopathological evaluation, none of the occasional gross lesions observed at necropsy were considered to be related to treatment with test iteml. No statistically significant difference in organ weight was found in male and female animals at any of the dose levels tested when compared to control. At histopathological evaluation, there were no abnormalities that could be attributed to treatment with the test items.

Conclusion

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with Propyl 4-hydroxybenzoate in male and femaleWistarrats, with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

The no observed adverse effect level (NOAEL) of Propyl 4-hydroxybenzoate in this study is determined at 1000 mg/kg body weight/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Guideline study according to GLP with a Klimisch rating of 1. Study design allows for endpoint related classification of substance

Justification for classification or non-classification

The available data on the repeated dose toxicity of the test substance does not meet the criteria for classification according to Regulation (EC) 1272/2008 , and is therefore conclusive but not sufficient for classification.