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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-28 till 2011-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 3 October 2008
Deviations:
yes
Remarks:
The rats were about 8-9 weeks old at the start of treatment in order to enable evaluation of the oestrus cycle (fertility parameters) during the last two weeks of the study. The deviations were considered not to have affected the validity of this study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
technical product

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The rats, 20 males and 20 females, were about 8-9 weeks old at the start of the treatment period (rationale: to enable evaluation of the oestrus cycle during the last two weeks of the study). The body weights at initiation of treatment were within ± 20% of the mean weight for each sex, and ranged from 235-286 g (mean 259 g) for males and from 165-196 g (mean 179 g) for females.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content, homogeneity and stability of the test substance in the carrier (tap water) were confirmed by HPLC-UV analysis.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 300, 1000 mg MPAAU/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Analysis of the dosing dilutions
Samples were taken from each dosing formulations prepared in the study. Analyses to determine the content, homogeneity and stability of the test substance in the carrier were conducted by HPLC-UV analysis (after storage in the animal room for approximately four hours and after storage in the refrigerator (2-10oC) for 7 days)

General clinical observations
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Neurobehavioural testing (arena testing, FOB and motor activity)
In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on all rats prior to the first exposure and then once weekly throughout the study. In week 4 of the study, the detailed clinical observations were included in the Functional Observation Battery (FOB and motor activity assessment were investigated in all rats in week 4 of the study).

Body weight
The body weight of each animal was recorded at initiation of treatment (day 0), and twice per week thereafter. In addition, the animals were weighed on their scheduled necropsy date after overnight fasting in order to calculate the correct organ to body weight ratios.

Feed consumption
Feed consumption was measured per cage by weighing the feeders. The consumption was measured over successive periods of 3 or 4 days throughout the treatment period. The results were expressed in g per animal per day.
Sacrifice and pathology:
Haematology
Haematology was conducted on all surviving animals. At necropsy, blood samples were taken from the abdominal aorta of the rats whilst under CO2/O2 anaesthesia. The rats were fasted overnight before necropsy (water was freely available). EDTA was used as anticoagulant. In each sample the following determinations were carried: haemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), reticulocytes, total white blood cells (WBC), differential white blood cells, prothrombin time, thrombocytes.
The following parameters will be calculated:
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

Clinical chemistry
Clinical chemistry was conducted on all surviving animals. At necropsy, blood samples were taken from the abdominal aorta of the rats whilst under CO2/O2 anaesthesia. The rats were fasted overnight before necropsy (water was freely available). The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The measurements listed below were made in the plasma: alkaline phosphatase activity (ALP) cholesterol (total), aspartate aminotransferase activity (ASAT) triglycerides, alanine aminotransferase activity (ALAT) phospholipids, gamma glutamyl transferase activity (GGT) creatinine, bilirubin (total) urea, bile acids inorganic phosphate, total protein calcium (Ca), albumin chloride (Cl), ratio albumin to globulin (calculated) potassium (K), (fasting) glucose sodium (Na).

Pathology
Gross necropsy
On day 28 of the study, after overnight fasting (water was freely available), the surviving animals were killed, in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. A thorough necropsy was also performed on male rat no.34 that died accidentally on day 22 of the study.

Organ weights
At scheduled necropsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights (g/kg body weight) were calculated based on the terminal body weight of the rats:
adrenals prostate brain seminal vesicles (with coagulating glands) epididymides spleen heart testes kidneys thymus liver uterus ovaries

Tissue preservation
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde.
adrenals oesophagus axillary lymph nodes ovaries brain pituitary* caecum prostate colon rectum duodenum seminal vesicles + coagulating glands epididymides 4 skeletal muscle (thigh) eyes spinal cord GALT (gut associated lymphoid tissue, spleen including Peyer's patches) sternum with bone marrow heart stomach ileum testes jejunum thymus kidneys thyroid liver trachea/bronchi lung urinary bladder mammary gland (females) uterus (with cervix) mandibular (cervical) lymph nodes* vagina mesenteric lymph nodes all gross lesions nerve-peripheral (sciatic)
The carcass containing any remaining tissues was also retained in formalin, but discarded after completion of the histopathological examination.

Histopathological examination
The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs listed above (except those marked with an asterisk) of all animals of the control group and the high-dose group, Gross lesions were examined in rats of all dose groups.
Other examinations:
Fertility parameters
Oestrus cyclicity
Vaginal smears to evaluate the oestrus cycle length and normality were made daily from day 15 until sacrifice on day 28. Smears were made and stained of all females, but evaluated only in the control and high-dose group.

Sperm analysis
Epididymal sperm motility, count and morphology
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups, and the motility was measured. The cauda epididymidis was dissected, weighed and thereafter minced in 3 ml M199 medium containing 0.5% BSA. Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups with the Hamilton Thorne Integrated Visual Optical System (IVOS).
In addition, a smear of the sperm solution was prepared and stained for all males, and two hundred spermatozoa of the smear of each male of the control group and high-dose group were analysed.

Testicular sperm count
At scheduled necropsy, the left testis of all males of all groups was placed on dry ice and subsequently stored in a freezer (<-70°) for later determination of the number of homogenization-resistant spermatids. Before analysis the testis was thawed and the tunica albuginea removed. After weighing, testicular parenchyma was minced and homogenized in 25 ml Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS in males of the control group and high-dose group. The daily sperm production was calculated as ‘number of spermatozoa per gram testicular parenchyma / 6.1’ (WF Blazak et al.; in Male Reproductive Toxicology, eds. RE Chapin and JJ Heindel, Methods in toxicology volume 3A, 1993).
Statistics:
Numerous tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Because numerous variables are subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results is based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
Fertility parameters
Estrus cyclicity: Smears obtained during the last 14 days prior to sacrifice of all control females and high-dose females were evaluated. The number of acyclic females, the mean length of the longest cycle, the number of cycles per animal and the number of females with a prolonged estrus period were comparable in both groups.

Sperm analysis
Epididymal sperm motility: Statistical analysis of sperm motility data indicated no significant differences between the test groups and the controls.
Epididymal sperm count: No statistically significant effects on epididymal sperm counts were observed.
Epididymal sperm morphology: No statistically significant effect on sperm morphology was observed.
Testicular sperm count: No effects on the number of spermatozoa per gram testicular parenchyma or on daily sperm production were observed.

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no indications of adverse effects of treatment

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect level (NOAEL) was placed at the highest dose level, 1000 mg MPAAU/kg body weight/day.
Executive summary:

In this sub-acute study, the safety of MPAAU was examined in Wistar rats. The test substance was administered as a solution in tap water by oral gavage to groups of 5 rats/sex, once daily during 28 consecutive days. The dose levels were 0 (tap water only), 50, 300 or 1000 mg MPAAU/kg body weight/day.

The administration of the test substance was well tolerated at all dose levels and did not induce any relevant changes in general condition, mortality, growth, feed intake, neurobehavioural observations, haematology, clinical chemistry, fertility parameters

or in macroscopic- and microscopic findings.

Because the administration of the test substance at levels up to 1000 mg/kg body weight/day did not induce any changes that were considered to be of toxicological significance, the no-observed-adverse-effect level (NOAEL) was placed at 1000 mg MPAAU/kg body weight/day.

Because adverse effects are not observed up to 1000 mg/kg of body weight/day the substance does not usually need to be assessed for long-term repeated dose toxicity.