Propyl 4-hydroxybenzoate

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propyl 4-hydroxybenzoate

Cas Number:

94-13-3

IUPAC Name:

Propyl 4-hydroxybenzoate

Test material form:

solid

Details on test material:

Composition:
Propyl 4-hydroxybenzoate: 99,7 %
4-Hydroxy-benzoic acid: 0.1 %
unspecified impurity: 0.2 %
Ethanol: < 200 ppm
Propanol: < 200 ppm

melting point: 98 °C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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).

 

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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).

Results and discussion

Results of examinations

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 %.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.