Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: 28d gavage test in rats including fertility parameters
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011-09-28 till 2011-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP and without deviations.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD 407 (Oct 2008), including fertility parameters
Deviations:
yes
Remarks:
The rats 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).
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
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.
Details on mating procedure:
no mating
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.
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)
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
50, 300, 1000 mg MPAAU/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
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.

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.
Oestrous cyclicity (parental animals):
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 parameters (parental animals):
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.
Litter observations:
no mating/offspring
Postmortem examinations (parental animals):
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).
Postmortem examinations (offspring):
no mating/offspring
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.
Reproductive indices:
no mating/offspring
Offspring viability indices:
no mating/offspring
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no indications of adverse effects of treatment on general condition or mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant changes in mean body weights.
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
ALAT activity was statistically significantly increased in females of the high-dose group. This finding occurred in one sex only and was not accompanied by corroborative changes in other parameters. There were no other statistically significant changes in clinical chemistry variables.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of MPAAU in rats.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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 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.
Reproductive performance:
not examined
Fertility parameters
Estrous 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 estrous 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.
Key result
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
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Conclusions:
In this sub-acute study, the safety of MPAAU was examined in Wistar rats (four groups of 5 males and 5 females each). The test substance was administered as a solution in tap water by daily oral gavage during 28 consecutive days, at levels of 0 (tap water only), 50, 300 or 1000 mg MPAAU/kg body weight/day. The dose volume was 10 ml/kg body weight/day in each group.
The content, homogeneity and stability of the test substance in the carrier were confirmed by analysis.
There were no adverse effects of treatment on general condition or mortality. One male rat of the high-dose group accidentally died on day 22 for reason unrelated to treatment. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance.
Body weights and feed intake were not affected by the administration of the test substance.
Haematology was conducted in all rats at necropsy. There were no treatment-related changes in red blood cell variables, clotting potential or in total and differential white blood cell counts. A slight increase in the absolute number (but not in percentage distribution) of basophils in females of the high-dose group was considered a chance finding.
Clinical chemistry, conducted in plasma obtained from all rats at necropsy did not show any treatment-related changes. An elevated ALAT activity in females of the high-dose group was not corroborated by changes in other parameters and considered a chance finding.
There were no treatment-related changes in absolute organ weights or in organ to body weight ratios.
Macroscopic examination at necropsy and microscopic examination of organs and tissues did not reveal adverse effects.
There were no effects of the test substance on fertility parameters (oestrus cyclus, epididymal sperm motility, sperm count and sperm morphology, and testicular spermcount and daily sperm production).
Because the administration of the test substance did not induce any changes that were considered to be of toxicological significance, the no-observed-adverse-effect level (NOAEL) was placed at the highest dose level, 1000 mg MPAAU/kg body weight/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study with Klimisch score 1 available, but no mating/no data in offspring available (=screening level).
Justification for non-submission of data from an OECD 416 test.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a repeated-dose 28-day oral (gavage) toxicity study (OECD 407) with MPAAU groups of 5 males and 5 females Wistar rats were exposed. The test substance was administered as a solution in tap water by daily oral gavage during 28 consecutive days, at levels of 0 (tap water only), 50, 300 or 1000 mg MPAAU/kg body weight/day. Because the administration of the test substance did not induce any changes that were considered to be of toxicological significance, the no-observed-adverse-effect level (NOAEL) was placed at the highest dose level, 1000 mg MPAAU/kg body weight/day. There were no effects of the test substance on fertility parameters (estrous cyclicity, epididymal sperm motility, sperm count and sperm morphology, and testicular spermcount and daily sperm production). - Estrous cyclus: 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 estrous 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. The NOAEL (toxicity to fertility) was placed at the limit dose, 1000 mg/kg bw/day.

In an oral (gavage) prenatal developmental toxicity test (OECD 414) with MPAAU pregnant NZW rabbits were dosed 0 (tap water only), 50, 300 or 500 mg MPAAU/kg body weight/day. On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14. The dams were sacrificed on GD 29 and macroscopically examined. Fetuses, placentas and reproductive organs were weighed. The fetuses were macroscopically examined and processed for visceral and skeletal examinations.

Signs of maternal toxicity were noted in the high-dose group and included mortality, conditional decline, blood around the perineum and/or in the cage, growth retardation and reduced feed intake. These changes were most evident in the initial phase of the study, but clinical signs, reduced growth and carcass weight, and lower feed intake remained after reduction of the high-dose level to 400 mg/kg bw/day.

Two high-dose rabbits had an early delivery and were necropsied (during partus) on GD 28.The gravid uterus weight tended to be lower in the high-dose group.The percentage of live fetuses per animal was decreased, and postimplantation loss and the number of (late) resorptions per animal were were increased in the high-dose group. Fetal weights were decreased in the mid- and high-dose group. These effects are deemed to be secondary to the marked maternal toxicity. The NOAEL (embryotoxicty) was placed at the mid-dose level, 300 mg/kg bw/day.

 

Based on evidence from OECD 414 rabbit embryotoxicity data and OECD 407 rat fertility data the adverse effects to reproduction are considered to be coincidental and/or secondary to general maternal toxicity. In absence of an appropriate 2-generation test in the rat the no observable adverse effect level of effects on fertility is taken from the study in the most sensitive species, the rabbit. The NOAEL for embryotoxicity (rabbit) = 300 mg/kg bw/day is based on lower gravid uterus weight, increased postimplantaion losses, decreased percentage of live fetuses per animal, and increased number of (late) resorptions found in high-dose animals.


Short description of key information:
OECD 407 including fertility parameters (rat): NOAEL (fertility) = 1000 mg/kg bw/day (limit dose).
OECD 414 (rabbit): NOAEL (embryotoxicity) = 300 mg/kg bw/day (increase in postimplantation loss and late resorptions coincidental/secondary to marked maternal toxicity in high-dose rabbits)
No data from OECD 416 test (waived) and no data from OECD 421/422 test (waived)

Justification for selection of Effect on fertility via oral route:
There were no effects of the test substance on fertility parameters (estrous cyclicity, epididymal sperm motility, sperm count and sperm morphology, and testicular spermcount and daily sperm production) in rats of a repeated-dose toxicity test at limit dose.

Effects on developmental toxicity

Description of key information

OECD 414 (rabbit): negative.

OECD 414 (rat): negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-23 till 2011-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP and without deviations. Study was conducted for a purpose other than REACH.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Ninety time-mated female New Zealand White rabbits of 4-5 months were obtained from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated females arrived at the testing facilities on GD 1 (23-25 November 2011 for Lot 1, 2 and 3, respectively). The rabbits were checked for overt signs of ill health and abnormalities upon arrival.
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.
Dilutions of the test substance in the vehicle were prepared weekly (namely on 25 November, 2 December, 5 December (only the adapted high-dose level), 9 December and 16 December 2011), and used within 9 days after preparation. The dilutions were stored in a refrigerator (2-10°C) in portions sufficient for one day. The vehicle for dosing the controls was similarly stored.
Details on mating procedure:
Ninety time-mated female New Zealand White rabbits of 4-5 months were obtained from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated females arrived at the testing facilities on GD 1 (23-25 November 2011 for Lot 1, 2 and 3, respectively).
Duration of treatment / exposure:
22 consecutive days
The test substance was administered once daily by oral gavage, as a dilution in tap water from GD 6 up to and including GD 28. Controls were treated with vehicle (tap water) only. The animals were dosed for the last time on the day prior to necropsy.
Frequency of treatment:
once daily
Duration of test:
Arrival of time-mated animals : 23-25 November 2011 (on GD 1)2
Start of the treatment : 28-30 November 2011 (from GD 6)
Date of scheduled necropsy : 21-23 December 2011 (GD 29)
No. of animals per sex per dose:
22 pregnant females/group
Details on study design:
On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14.
Maternal examinations:
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. 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.
Animals showing signs of severe intoxication, particularly if death appeared imminent, were humanely killed to prevent loss of tissues by autolytic degeneration.

Body weight: The body weight of each animal was recorded on GD 1, 6, 9, 12, 15, 18, 21, 24 and 29.

Feed consumption: The feed consumed by each mated female was measured over the periods: GD 1-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-29 by weighing the feeders.

Water consumption: Water consumption was not measured.

Necropsy: The study was terminated with the necropsy of the rabbits on GD 29 (on 21, 22 and 23 December 2011 for the rabbits of lot 1, 2 and 3, respectively). Two high-dose rabbits, who had an early delivery were necropsied during partus on GD 28¹.
The rabbits were killed by an intravenous injection in the ear of sodium pentobarbital and examined for gross abnormalities. Fetuses were killed by hypothermia. Any dam found dead or killed in extremis during the study was also examined macroscopically, and the number of corpora lutea and implantations were recorded. Maternal tissues showing severe macroscopic abnormalities and the kidneys were sampled and fixed in a neutral, aqueous phosphate buffered 4% solution of formaldehyde for possible future microscopic examination.

¹ Rabbit no 149 (lot 2) had an early delivery and was necropsied (during partus) on 21 December 2011.
Rabbit no 167 (lot 3) had and early delivery and was necropsied (during partus) on 22 December 2011.
Ovaries and uterine content:
The uteri (including the fetuses), ovaries and placentas of all females killed at the end of the study were examined for the following parameters:
- number of corpora lutea
- number of implantation sites
- number of early and late resorptions
- number of live and dead fetuses
- number of grossly visible malformed fetuses and fetuses with external
abnormalities
- weight of ovaries
- weight of uterus, containing placentas and fetuses
- weight of uterus, empty
- weight of fetuses
- weight of the placentas
- gross evaluation of the placentas.


Fetal examinations:
Fetopathological examination:
All fetuses of all litters were examined by careful dissection for visceral abnormalities, but visceral examination of the head was conducted in only half of the fetuses in each litter (the head of the other fetuses was subjected to skeletal evaluation, see below). During the visceral examination, the sex of the fetuses was determined.
To examine the head for visceral abnormalities, half of the number of foetuses in each litter was decapitated and their heads fixed in Bouin’s fixative and subsequently free-hand sectioned according to Van Julsinga and Bennett (1977). The sections obtained were examined for visceral abnormalities.
After the visceral examinations, the intact and decapitated bodies were eviscerated, fixed in 70% alcohol, cleared in potassium hydroxide and stained with Alizarin Red S. All skeletons of each group were examined for skeletal abnormalities and then retained. The skull was examined in only half of the fetuses of each litter (the head of the other fetuses was subjected to visceral evaluation, see above). During the fetopathological examinations the observer was unaware of the group of the fetuses.
Statistics:
Statistical analysis of the results:

The data were analysed using the methods mentioned below.
Clinical findings, mortality data, number of pregnant females, females with live fetuses, parental necropsy observations and the fetopathological screening: Fisher's exact probability test.
Body weight, body weight gain, organ weights and feed consumption data: one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
Number of corpora lutea, implantations, live and dead embryos or fetuses and early and late resorptions: Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test.
Statistical evaluations on variables associated with the fetuses were considered on a litter basis in accordance to standard procedures. The statistics applied are indicated in the tables.
Results were taken as statistically significant where the probability of the results was <0.05 or <0.01. The final interpretation of results was based not only on statistical analysis but also on other considerations such as nature of the finding, dose-response relationships and whether the results were significant in the light of other biological and pathological findings.
Indices:
Reproductive performance:

For each group the following data were presented:
- number of females mated
- number of females pregnant
- number of females with no viable fetuses
- number of females with live fetuses
- number of corpora lutea
- number of implantation sites
- number of live fetuses
- number of dead fetuses
- number of live male fetuses
- number of live female fetuses
- number of early/late/total number of resorptions.

For each group the following indices were calculated:
- female fecundity index = (number of pregnant females/number of females mated) x 100
- pre-implantation loss = [(number of corpora lutea - number of implantation sites)
/ number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live fetuses)
/ number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Summary of parental data
Maternal toxicity were noted in the high-dose group and included:
- Mortality; one rabbit of the high-dose group was found dead on GD 9. Two other rabbits of this group were killed in extremis on GD 13 and GD 21.
- Conditional decline; 10 rabbits in the high-dose group showed blood around the perineum and/or in the cage.
- Weight loss or growth retardation, and reduced feed intake were noted in the high-dose group.
These changes were most evident in the initial phase of the study, but remained after reduction of the high-dose level to 400 mg/kg bw/day.
No treatment-related macroscopic findings of the dams were observed at Caesarean section. The carcass weight was reduced in the high-dose group. Two high-dose rabbits had an early delivery and were necropsied (during partus) on GD 28.
- Feed intake also tended to be reduced in the mid-dose group, but the differences with the controls were not statistically significant.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Reproduction data
A number of 22 mated females per group resulted in 21, 20, 18 and 17 litters for the control, low-dose, mid-dose and high-dose group, respectively. Two high-dose rabbits (no’s 149 and 167) had an early delivery and were necropsied (during partus) on GD 28. Results obtained in these two rabbits are not included in the mean data (tables), but all data are shown in the individual data (appendices).
The percentage of live fetuses per animal was statistically significantly decreased in the high-dose group. Postimplantation loss and the number of resorptions per animal were increased in this group. The latter finding was statistically significant and mainly due to an increase in late resorptions.

Fetal placental findings
Fetal placental findings were not affected by the treatment.

Fetal and placental weights
Fetal weights were decreased in the mid- and high-dose group. The differences with the controls were statistically significant for all fetuses and for male fetuses. Fetal weights were also relatively low in the low-dose group. The latter finding was not statistically significant, and, moreover, due to the higher number of fetuses per animal in this group. Therefore the changes in fetal weights in the low-dose group are not considered to be treatment-related.
Placental weights were not affected by the treatment.

Fetal external observations
There was one fetus in the mid-dose group (from dam no.105) showing a malformed head (namely agenesis of all soft tissues and all skull bones, except tongue and lower jaw). One fetus, from a high-dose dam (no. 149) with an early delivery, showed absence of the cranial vault. Umbilical hernia was observed in one fetus of the low-dose and the mid-dose group, and a small fetus was noted in the low-dose and the high-dose group.
Because there was no dose-response relationship, the latter findings were not ascribed to treatment.

Visceral examination

Visceral malformations
One fetus in the mid-dose group (from dam no.105) showed agenesis of all soft tissues and all skull bones, except tongue and lower jaw . The umbilical hernia, observed in one fetus of the low-dose and the mid-dose group was associated with lobular torsion of the liver. Retrocaval uterus was an incidental finding noted in two control fetuses and one low-dose fetus. No other visceral malformations were observed.

Visceral anomalies
No statistically significant or treatment-related differences in the incidences of visceral anomalies were observed among the groups.

Visceral variations
The incidences of fetuses and litters showing a lobular appendix of the gallbladder were statistically significantly increased in the high-dose group.
Four fetuses of the high-dose group showed cysts in the ovaries. This finding was statistically significant, but because it involved only one litter it is probably incidental. Other incidental findings were statistically significant decreased incidences of haemorrhagic areas in the uterus (low- and mid-dose group), and haemorrhagic fluid in the pericard and abdomen (high-dose group). No other statistically significant visceral variations were observed.

Skeletal examination

Skeletal malformations
One fetus in the mid-dose group (from dam no.105) showed agenesis of all skull bones and all soft tissues, except lower jaw and tongue. One fetus, from a high-dose dam with an early delivery (no. 149), showed agenesis of frontal, parietal and supra occipital skull bones. Acephaly has been observed, though at a low incidence, in previous studies conducted in our laboratory with this strain of rabbits without a correlation with treatment. Because of the absence of similar or correlated findings in any other animal in this study, the present findings are not ascribed to treatment.
Other skeletal malformations included the ribs, sternebrae, vertibral column, thoracal bodies and arches. They were equally divided among the various groups and/or present in one or a few fetuses. Because no remarkable or statistically significant differences between the treatment groups and the controls were observed in the incidences of these findings, they were not ascribed to treatment.

Skeletal anomalies
No statistically significant differences between the treatment groups and the controls were observed in the incidences of skeletal anomalies.

Skeletal variations
The incidence of fetuses showing an irregular shape of one strenebra was slightly, though statistically significantly increased in the high-dose group. This finding is probably a chance finding because the incidence of irregular shape of ‘two or more’ strenebrae was not affected.
No other statistically significant differences between the treatment groups and the controls were observed, except for an incidental decrease in fetuses showing accessory lumbar rib(s) in the mid-dose group.

Skeletal retardations
Various statistically significant differences were observed in incidences of retardations in ossification, namely:
- Sternebrae: The incidence of fetuses showing one unossified sternebra was increased in the high-dose group.
- Coracoids: The incidence of fetuses showing unossified coracoid was increased in the mid- and high-dose group. An incidental decrease was noted in incidence of incompletely ossified coracoid in the low-dose group.
- Metacarpals: The incidence of fetuses showing 1-2 unossified metacarpals was increased in the mid-dose group, but this finding was not confirmed in the high-dose group.
- Phalanges: The incidence of fetuses showing 1-4 incompletely ossified medial digits of the frontal phalanges was increased in the high-dose group.
In the low-dose group, the incidence of fetuses with 1-4 unossified medial digits of the frontal phalanges was increased. This finding was, however, not confirmed by dose-related or statistically significant changes in the higher-dose groups and is therefore considered a chance finding.
- Epiphyses of the femur: The incidence of fetuses of which the distal epiphysis of the femur was unossified was increased in the mid- and high-dose group.
- Epiphysis of the tibia: The incidence of fetuses of which the proximal epiphysis of the tibia was unossified was increased in the mid- and high-dose group.
An incidental decrease in incompletely ossified proximal epiphysis of the tibia was observed in the fetuses of the low-dose group.
- Epiphysis of the humerus: The incidence of fetuses of which the proximal epiphysis of the humerus was unossified was increased in the mid- and high-dose group. The incidence of fetuses with the distal epiphysis of the humerus unossified, was increased in the low- and mid-dose group. This finding was, however, not confirmed by dose-related or statistically significant changes in the high-dose group and is therefore considered a chance finding.
As a result of the above retardations in ossification, the total incidence of fetuses showing skeletal retardations was increased in the mid- and high-dose group.


Summary of reproduction data
- No differences were noted in the female fecundity and gestation indices. No effects on the number of corpora lutea, implantation sites, live and dead fetuses, sex ratio, placenta or placental weights were observed.
- The gravid uterus weight tended to be lower in the high-dose group.
- The percentage of live fetuses per animal was decreased and postimplantation loss and the number of (late) resorptions per animal were increased in the high-dose group.
- Fetal weights were decreased in the mid- and high-dose group.


Summary of fetal examination
One fetus of a high-dose dam that had an early delivery showed absence of the cranial vault (agenesis of frontal, parietal and supra occipital skull bones). A fetus of a mid-dose dam showed a malformed head (agenesis of all soft tissues and all skull bones, except tongue and lower jaw). These findings were considered incidental and not ascribed to treatment. There were no other remarkable visceral and skeletal malformations or anomalies. However, a visceral variation (increased incidence of a lobular appendix of the gall bladder) was noted in the high-dose group, while several skeletal retardations in ossification were observed in the mid- and the high-dose group.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Discussion and conclusion

The objective of this study was to provide data on the effects of MPAAU on pregnant New Zealand White rabbits and the development of the embryo and fetus following daily oral administration by gavage during gestation days (GD) 6-28.

The dose levels were 0 (tap water only), 50, 300 or 500 mg MPAAU/kg body weight/day. On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14. The dams were sacrificed on GD 29 and macroscopically examined. Fetuses, placentas and reproductive organs were weighed. The fetuses were macroscopically examined and processed for visceral and skeletal examinations.

 

Signs of maternal toxicity were noted in the high-dose group and included mortality, conditional decline, blood around the perineum and/or in the cage, growth retardation and reduced feed intake. These changes were most evident in the initial phase of the study, but clinical signs, reduced growth and carcass weight, and lower feed intake remained after reduction of the high-dose level to 400 mg/kg bw/day.

 

Two high-dose rabbits had an early delivery and were necropsied (during partus) on GD 28. The gravid uterus weight tended to be lower in the high-dose group. The percentage of live fetuses per animal was decreased, and postimplantation loss and the number of (late) resorptions per animal were were increased in the high-dose group. Fetal weights were decreased in the mid- and high-dose group.

 

Two remarkable malformations were noted at necropsy. One was the absence of the cranial vault (agenesis of frontal, parietal and supra occipital skull bones) in a fetus of a high-dose dam that had an early delivery. The other was a malformed head (agenesis of all soft tissues and all skull bones, except tongue and lower jaw) in a fetus of a mid-dose dam. Acephaly has been observed, though at a low incidence, in previous studies conducted in our laboratory with this strain of rabbits without a correlation with treatment. Because of the absence of similar or correlated findings in any other animal in this study, the present findings are not ascribed to treatment.

 

There were no other noticeable visceral and skeletal malformations or anomalies.

However, a visceral variation (increased fetal and litter incidence of a lobular appendix of the gall bladder) was noted in the high-dose group.

In addition, several skeletal retardations in ossification were observed in the mid- and/or the high-dose group, namely unossified sternebra (high-dose group), unossified coracoid (mid- and high-dose group), 1-4 incompletely ossified medial digits of the frontal phalanges (high-dose group), unossified distal epiphysis of the femur and proximal epiphysis of the tibia (mid- and high-dose group). These retardations in ossification are generally considered to be transient and deemed to be secondary to reduced maternal feed intake and reduced maternal body weight in these groups.

Conclusions:
Marked maternal toxicity were observed in the high-dose group, and the maternal no-observed-adverse-effect level (NOAEL) was 300 mg MPAAU/kg body weight/day.

No visceral or skeletal malformations or anomalies were observed at any dose level that are treatment related. Effects on reproductive parameters were noted in the high-dose group, consisting of increased postimplantation loss and decreased percentage of live fetuses per animal. A visceral variation was noted in the high dose group. In the mid- and high-dose group, fetal weights were decreased and skeletal retardations in ossification were observed.

No frank malformation were observed. The visceral variation in the high dose group, and the skeletal retardations in ossification in high and mid dose groups are considered transient in nature and deemed to be secondary to reduced maternal feed intake and the overall growth retardation in these groups. Therefore, the results indicate that MPAAU was not teratogenic in the NZW rabbit. The developmental no-observed-adverse-effect level (NOAEL) was 500 mg MPAAU/kg body weight/day.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-11 until 2019-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as GD ‘0’.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week between GD 5 until GD 19.
Frequency of treatment:
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Duration of test:
until GD 20
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
mid dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
25 females/dose group
Control animals:
yes, concurrent vehicle
Maternal examinations:
Body Weight and Food Consumption
All animals were weighed once before initiation of pairing to ensure that the body weights are within ± 20 % variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.
Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. 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.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.
Ovaries and uterine content:
Post-Mortem Examination
On gestation day 20, sperm positive (presumed pregnant females) were subjected to a caesarean section after sacrificing the animals using anesthesia (e.g. ketamine/xylazine).
At the time of termination or death during the study, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4 % neutral-buffered formaldehyde.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appeared non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.
Thyroid/parathyroid glands from all dams were preserved in 4 % neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or fetal deaths as well as the number of viable fetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of fetuses in each uterine horn was also recorded.
Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.

Evaluation of Thyroid Hormones
Thyroid hormone levels from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site and were stored under appropriate conditions. Blood samples were assessed (one measurement per animal) for serum levels of thyroid hormones and pituitary-thyroid axis hormones (T3, T4, TSH). Hormone determination were performed on the MagPIX, Luminex or on the DRG:HYBRID-XL Analyzer, DRG.

Histopathology
A full histopathology was carried out on the preserved thyroid gland from all dams which were sacrificed at the end of the treatment period.
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.
Fetal examinations:
Fetal Evaluations
All fetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance (AGD). Each fetus was examined for external anomalies and the AGD of each fetus was measured. Fetal body weight measured on GD 20 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of fetus weight).
Particular attention was paid to the reproductive tract which was examined for signs of altered development. External fetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent/cryptorchidism was noted in male fetuses.

One half of each litter were examined for soft tissue anomalies of the body by a microdissection technique. After the completion of the external examination, fetuses were transferred to plastic bottles containing Bouin’s solution for later soft tissue/visceral and craniofacial examination (internal structures including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique). The soft tissue/visceral evaluation of the body and craniofacial examination of the heads was performed of the same fetuses of 20 litters per group. The remaining fetuses were processed by Alizarin staining and 20 litters per group were examined for skeletal alterations.

External Examination
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.
Visceral Examination
Fetuses scheduled for the visceral examination were pinned with the ventral side up under a stereo microscope. The abdominal and thoracic cavities of all fetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off centre and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.
The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart were recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.
After the completion of the visceral examination by the microdissection technique, fetuses were transferred to plastic bottles containing Bouin´s solution and 95% ethanol after completion of three weeks for later craniofacial examination by the razor-blade-serial-section technique.
Craniofacial Examination
A single fetus was decapitated and the head of the fetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.

Skeletal Examination
Fetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95 % ethanol. These fetuses were processed using the Alizarin red staining technique. After fixation in 95 % ethanol, the fetuses were macerated with a 1 % aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025 % in 1 % aqueous potassium hydroxide) for 1 day. After that the fetuses were again transferred to 1 % KOH. Alizarin stained fetuses were then cleared and dehydrated in a solution containing 2 parts of 70 % ethanol, 2 parts of glycerine and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerine.
The stained fetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centres. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centres and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each fetus.
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed 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. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Indices:
The findings of this study were evaluated in terms of the observed effects.
Maternal and fetal test results, including an evaluation of the relationship between the exposure of the animals to the test substance and the incidence and severity of all findings as well as findings of fetal external, soft tissue and skeletal anomalies are reported.
Body Weight and food consumption was presented for intervals during gestation days 0-5, 5-8, 8-11, 11-14, 14-17 and 17-20.
The number of live births and post implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, live and dead fetuses and resorptions, AGD, litter size and litter weights were summarized in tabular form.
The relative weights of thyroid/parathyroid glands were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.
All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Analytical results and histopathological findings are presented in separate phase reports in the Annexure of this report.
Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.3.4, Pathology Data Systems Ltd.).
Historical control data:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation and moving the bedding observed in the MD and HD group were seen transiently and were considered as slight clinical signs elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to the oral administration, but not systemic toxicity. Other slight clinical signs like hairless areas, scratches/cuts and piloerection observed in single animals followed no dose-dependency and were not considered toxicologically relevant.
No signs of abortion or premature delivery prior to the scheduled sacrifice were observed in any of the females.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in any of the test item-treated groups or the control group during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant or statistically significant effects on body weight, body weight development and food consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Maternal thyroid hormone (T3, T4, TSH) levels showed no toxicologically relevant effects.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No biologically relevant or statistically significant differences were observed in mean maternal terminal body weight, gravid uterus weight and resulting adjusted maternal weight of test item-treated females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No relevant or statistically significant effects were observed on prenatal data including the number of corpora lutea, implantation sites, live fetuses, early and late resorptions, pre- and post-implantation loss and sex ratio.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In correlation with no statistically significant differences in thyroid/parathyroid weight and macroscopic observation, histopathological evaluation revealed no test item-related morphological lesions in the thyroid gland. No test item-related necropsy findings were observed up to the dose of 1000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Successful mating resulted in 24/25 pregnancies in the LD group, MD group and HD group, each, compared to 23/25 pregnancies in the control group. No signs of abortion or premature delivery prior to the scheduled sacrifice were observed in any of the females.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No relevant or statistically significant effects were observed on prenatal data including the number of corpora lutea, implantation sites, live fetuses, early and late resorptions, pre- and post-implantation loss and sex ratio.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed in any of the test item-treated groups or the control group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: No effect found
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal weight on a per litter basis showed no statistically significant effects in test item-treated animals. Statistically significantly higher mean fetal weight (on a fetal basis) of male and female fetuses of the MD group and male fetuses of the HD group were not considered toxicologically relevant due to the slightness of the difference and the lack of dose-dependency.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No relevant or statistically significant effects were observed on prenatal data including the number of corpora lutea, implantation sites, live fetuses, early and late resorptions, pre- and post-implantation loss and sex ratio.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external abnormalities considered to be of toxicological relevance were observed in any of the test item-treated groups. Statistical analysis of fetal and litter incidences showed no significant differences compared to the control group. Single findings (absent lower jaw, mouth, tongue and misshapen head or absent lower jaw and an open eye in two different fetuses of the MD group and haematoma in the control and MD group) were considered incidental due to the lack of consistency between the test item-groups.

Testicular descent was observed to be complete in all examined male fetuses. No effect of the test item on testicular descent was evident in test item-treated male fetuses when compared to male control fetuses.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no craniofacial abnormalities of toxicological relevance in any of the test item-treated groups. Statistical analysis of fetal and litter incidences showed no significant differences compared to the control group. Single findings including subcutaneous haematoma, a large or misshapen cerebrum, dilated ventricles, perimeningal space filled with red material, an increased perimeningal space were observed without dose-dependency and were not related to treatment with the test item.

In the skeletal examination incomplete ossification was seen in several bones of all groups including the control group as usual for fetuses on gestation day 20. Values showed no dose-dependent pattern or litter incidences for ossification status were statistically insignificant between the dose groups and controls and thus findings concerning the ossification were not considered test item-related. Statistically significant higher fetal incidence of incomplete ossification of caudal arches in the LD group and statistically significantly higher fetal incidence of unossified caudal arches in the LD and MD group were not considered test item-related as no dose-dependency and no differences in the HD group were observed. Statistically significantly higher fetal incidence in the HD group and a dose-dependent trend in LD and MD group was observed for rudimentary thoracolumbal ribs. However, this observation was not considered adverse due to the low number of incidences. The findings of full supernumerary thoracolumbal ribs, rudimentary cervical ribs, wavy ribs, pelvic girdle caudal shift, misaligned ossified/split/fused sternebra, bipartite or irregular ossification of thoracic centra and misshapen/bent/short scapulae were observed in all groups without statistical significance or dose dependency and thus were not considered test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Internal observation of the fetal viscera revealed a range of findings in all test item-treated groups and the control group. Affected organs/tissues were the umbilical artery, stomach, liver, pancreas, testis, azygos vein, thyroid gland, lung lobe, spleen and the thymus. Abdominal haemorrhages and subcutaneous haemorrhages were observed in all dose groups and the control group. However, these findings were not considered test item-related as litter incidences showed no statistically significant differences in dose groups compared to the control group and findings were observed without any dose-dependent trend. Statistically significantly higher mean fetal incidence (on a litter basis) of a discoloured pancreas in the MD group was considered incidental as LD and HD group were not affected. Testicular descent was observed to be complete in all examined male fetuses. No effect of the test item on testicular descent was evident in test item-treated male fetuses when compared to male control fetuses.
Other effects:
no effects observed
Description (incidence and severity):
Mean absolute and relative AGD was significantly statistically higher in males of the MD group and females of the MD and HD group. However, this may be related to the slightly higher mean fetal weight in these groups and without any dose-dependency it was not considered toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: No effect found.
Key result
Developmental effects observed:
no
Conclusions:
On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with MPAAU at dose levels of 100, 300 and 1000 mg/kg body weight/day administered by oral gavage on GD 5 to 19, the following conclusions can be made:
No mortality was observed during the treatment period of this study. All clinical signs observed in terminally sacrificed females were incidental or non-adverse in nature.
No treatment-related effect considered of toxicological relevance were observed on body weight development, food consumption, prenatal data parameters, litter data parameters and pathology of terminally sacrificed females up to highest tested dose of 1000 mg/kg bw/day.
No treatment-related and toxicologically relevant external, visceral, craniofacial or skeletal findings were observed in any of the test item-treated groups. Few skeletal findings including incomplete ossification of bones and other skeletal findings showed no dose-dependency or relevant statistical significance and thus were not considered to be adverse effects caused by treatment with the test item.
No adverse systemic effects of the test item were found up to 1000 mg/kg body weight/day. Thus, the NOAEL for both maternal toxicity and fetal toxicity in this study is considered to be 1000 mg/kg body weight/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Two GLP studies (in rodent & non-rodent species) with Klimisch score 1 are available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

On the basis of a prenatal developmental toxicity study in Wistar rats with MPAAU at dose levels of 100, 300 and 1000 mg/kg body weight/day administered by oral gavage on GD 5 to 19, the following conclusions can be made:

No mortality was observed during the treatment period of this study. All clinical signs observed in terminally sacrificed females were incidental or non-adverse in nature.

No treatment-related effect considered of toxicological relevance were observed on body weight development, food consumption, prenatal data parameters, litter data parameters and pathology of terminally sacrificed females up to highest tested dose of 1000 mg/kg bw/day.

No treatment-related and toxicologically relevant external, visceral, craniofacial or skeletal findings were observed in any of the test item-treated groups. Few skeletal findings including incomplete ossification of bones and other skeletal findings showed no dose-dependency or relevant statistical significance and thus were not considered to be adverse effects caused by treatment with the test item.

No adverse systemic effects of the test item were found up to 1000 mg/kg body weight/day. Thus, the NOAEL for both maternal toxicity and foetal toxicity in this study is considered to be 1000 mg/kg body weight/day.

 

In an oral (gavage) prenatal developmental toxicity test (OECD 414) with MPAAU pregnant NZW rabbits were dosed 0 (tap water only), 50, 300 or 500 mg MPAAU/kg body weight/day. On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14. The dams were sacrificed on GD 29 and macroscopically examined. Foetuses, placentas and reproductive organs were weighed. The foetuses were macroscopically examined and processed for visceral and skeletal examinations.

Signs of maternal toxicity were noted in the high-dose group and included mortality, conditional decline, blood around the perineum and/or in the cage, growth retardation and reduced feed intake. These changes were most evident in the initial phase of the study, but clinical signs, reduced growth and carcass weight, and lower feed intake remained after reduction of the high-dose level to 400 mg/kg bw/day. The maternal no-observed-adverse-effect level (NOAEL) was 300 mg MPAAU/kg body weight/day.

No visceral or skeletal malformations or anomalies were observed at any dose level that are treatment related. Effects on reproductive parameters were noted in the high-dose group, consisting of increased postimplantation loss and decreased percentage of live foetuses per animal. A visceral variation was noted in the high dose group. In the mid- and high-dose group, foetal weights were decreased and skeletal retardations in ossification were observed.

The visceral variation in the high dose group, and the skeletal retardations in ossification in high and mid dose groups are considered transient in nature and deemed to be secondary to reduced maternal feed intake in these groups.  The no-observed-adverse-effect level (NOAEL) for developmental toxicity was 500 mg MPAAU/kg body weight/day.

 Justification for selection of effect on developmental toxicity: via oral route, two reliable studies are available.

Justification for classification or non-classification

Based on the available data the substance is not hazardous to reproduction and a classification according to EC 1272/2008 is deemed to be not necessary.