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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-12-03 until 2019-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
measured concentrations
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
measured concentrations
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
measured concentrations
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

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 period.
Food consumption was measured weekly during the treatment period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. Inadvertently, clinical observations were not made on one day (25 February 2019, study day 74, 75, 76 or 77 respectively (staggered start)).
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. 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 at least once a week thereafter.
Ophthalmological examination using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

Functional Observations
Once before the first exposure and once in the last week multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests [14]. These tests were conducted in all animals.
Sacrifice and pathology:
Haematology
Haematological parameters were examined at the end of the treatment 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 (HGB)
red blood cell count (RBC)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin (MCH)
mean corpuscular haemoglobin concentration (MCHC)
reticulocytes (Ret)
platelet count (PLT)
white blood cells (WBC)
neutrophils (Neut)
lymphocytes (Lym)
monocytes (Mono)
eosinophils (Eos)
basophils (Baso)
large unstained cells (Luc)

Blood Coagulation
Coagulation parameters were examined at the end of the treatment 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 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 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 prior to or as part of the sacrifice of the animals. Additionally, urine colour / appearance were recorded. The following parameters (Table 7) were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).
specific gravity
nitrite
pH-value (pH)
protein
glucose
ketone bodies
urobilinogen (UBG)
bilirubin (BIL)
erythroctes (Ery)
leukocytes (Leuc)

Pathology
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anaesthesia (ketamine/xylazine) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.

Organ Weight
The wet weight of the organs (Table 8) was taken from sacrificed animals as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of the animal euthanised on study day 8 for animal welfare reasons were not recorded:
liver, uterus with cervix
kidneys, thymus
adrenals, thyroid / parathyroid glands (were weighed after fixation)
testes, spleen
epididymides, brain
prostate, seminal vesicles and coagulating glands, pituitary gland
ovaries, heart
The following tissues 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.
adrenal glands x x
all gross lesions x x
aorta x x
brain (incl. medulla/pons, cerebellar and cerebral
cortex) x x
caecum x x
colon x x
duodenum x x
epididymides x x
eyes with optic nerve and Harderian gland x --
femur with knee joint x --
heart x x
ileum (including Peyer´s patches) x x
jejunum x x
kidneys x x
liver x x
lungs x x
lymph nodes (mandibular) x --
lymph nodes (mesenteric and axillary) x x
mammary gland area (male and female) x x
oesophagus x x
ovaries x x
oviducts x --
pancreas x x
pituitary x x
prostate and seminal vesicles with coagulating glands as a whole x x
rectum x x
salivary glands (sublingual, submandibular, parotis) x x
sciatic nerve x x
skeletal muscle x x
skin x x
spinal cord (cervical, thoracic and lumbar segments) x x
spleen x x
sternum (with bone marrow) x x
stomach x x
testes x x
thymus x x
thyroid gland including parathyroid glands x x
tongue x --
trachea x x
ureters x --
urinary bladder x x
uterus with cervix and vagina x x
The animal intercurrently euthanised for animal welfare reasons was also subjected to a gross necropsy and the organs preserved for a histopathological examination.

Histopathology
The afore-listed organs (Table 9) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in the high dose group.
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.
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. 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):
In the female HD group, prone position was noted on treatment days 68 to 73 for one animal and dehydration on day 15 for five animals. As these findings were observed in one single animal or on one single day, a toxicological effect of the test item is not considered.
The findings crust and/or hairless area at the cheek, head or forelimbs in several female animals of the control group or for one female LD group (hairless area at forelimbs, hindlimbs, lateral area, abdomen, legs) are not considered to be of biological or toxicological relevance.
Moving the bedding and/or slight to moderate salivation (in one HD female animal with a severe grade) were noted in 4 out of 10 male LD group animals and in all male and female MD and HD group animals. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore considered to be signs of a local reaction after test item treatment rather than a systemic adverse effect.
Regarding detailed clinical observation, statistical significance was noted for males in week 3 for a slightly increased mean score of salivation in the HD group (15 % above control), in week 12 for animal sleeps (87.1 % below control) and animal moving in cage (305 % above control) in the MD group. In females, statistical significance was found in week 2 for an increase of 10% above control for response to handling in the LD group, for an increase of salivation in the HD group in week 3 (20 % above control), in week 4 (30 % above control), in week 5 (25 % above control), in week 6 (25 % above control), in week 8 (20 % above control), in week 10 (35 % above control) and additionally in week 11 (30 % above control) and in the HD group for an increased mean score of animal moving in cage in week 6 (100 % above control). The statistically significant effects on detailed clinical examination parameters were not considered to be test item-related as they were seen occasionally in single weeks. Significantly increased salivation, which was observed in several weeks in the female HD group, was assessed under consideration of the presence of salivation during daily clinical observation and therefore not considered to be a systemic adverse effect of the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control male animal (animal no. 4) was euthanized in moribund condition on study day 8. Clinical findings on day of sacrifice were prone position, severely reduced spontaneous activity, eyes closed and abnormal breathing. No clinical findings were seen on treatment days 1 to 7. Considering histopathological assessment, death of animal no. 4 was considered to be incidental. The cause of morbidity was not evident at the histopathological evaluation and is assumed to be related to gavage procedure. There were no mortalities in the remaining males and females of the control and all dose groups until the end of the treatment period.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects were found for all haematological and coagulation parameters of all male and female dose groups at the end of the treatment period.
A statistical significant decrease of 71.4 % below control was found for the mean value of eosinophils in the male MD dose group and for an increase of monocytes in the female MD dose group (51.1 % above control). The statistical significant changes from control group were found without dose dependency and therefore the single findings are not considered to be of toxicological relevance.
No statistical significances were noted for coagulation parameters in any male and female dose group when compared to control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test item had no toxicologically relevant effects on parameters of clinical biochemistry or hormone analysis of males and females in any of the test item-treated groups.
Statistical significance was found in the female HD group for a decrease of 64.87 % below control for total bile acid. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency. Additionally, no test item- related effects were noted at histopathological assessment. Therefore, the statistical significance is considered to be of no biological or toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no adverse effect of the test item on urinary parameters measured at the end of the treatment period.
Behaviour (functional findings):
effects observed, non-treatment-related
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 for the male and female groups.
The statistical significant mean scores (decrease of animal sleeps, increase of moving in cage) for the male LD group in the last week of treatment were not considered to be an effect of test item treatment as they were seen without dose dependency.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Treatment with the test item had no toxicologically relevant effects on absolute and relative organ weight of males and females in any of the test item-treated groups. No statistical significant organ weight changes were found for all male and female dose groups when compared to control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic finding of the male control animal sacrificed in moribund condition was a dark red coloured right caudal lobe of the lung. Considering histopathological assessment, the cause of morbidity was not evident at the histopathological evaluation. In one HD male animal, a cyst at the prostate gland and spotted harderian glands were observed at necropsy on study day 91. Regarding the female HD group, necropsy findings were few black foci on the stomach and red coloured axillary or mandibular lymph nodes. Uterus dilatation was found in one control and one MD animal and several HD animals. Considering histopathological evaluation, all gross lesions observed at necropsy for terminally sacrificed animals were deemed to be incidental or represented the normal animal physiology and therefore, considered to be not related to the treatment with the test item.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were observed during the histopathological evaluation. All findings recorded in decedent and surviving animals were deemed to be incidental or were within the range of background alterations that may be recorded in animals of this strain and age and in this study type.
Considering histopathological assessment, there were no organ weight changes, gross lesions or histological alterations that could be attributed to the treatment with the test item. Thus, the histopathological NOEL (no observed effect level) could be established at 1000 mg/kg bw.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no relevant effect found

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) of the test item MPAAU in this study is considered to be 1000 mg/kg body weight/day.
Executive summary:

MPAAU was assessed in a 90-Day Repeated Dose Oral Toxicity study in male and female Wistar rats, with dose levels of 100, 300 and 1000 mg/kg body weight/day. No test-item related mortality was observed and no adverse effects of the test item were found for male and female clinical observations, functional observations, body weight development, food consumption, haematology and coagulation, clinical biochemistry and hormone analysis, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treated dose groups.

Hence, the no observed adverse effect level (NOAEL) of the test item MPAAU in this study is considered to be 1000 mg/kg body weight/day.