Methylphosphonic acid, compound with amidinourea (1:1)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Justification of non-submission of data

The evidence form repeated dose studies with MPAAU in rats (OECD 407, 408, 414), each with NOAEL = 1000 mg/kg bw/day, support the waiver for the data requirement of an OECD 443 study: 

The extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reproductive effects observed:

not specified

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

Stricker (2020)/supp/Prenatal developmental toxicity study in rats with hormone analysis

Type of information:

experimental study

Adequacy of study:

supporting 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

Guideline:

other: OECD 414

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Vehicle:

water

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’.

Analytical verification of doses or concentrations:

yes

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.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

LD

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

MD

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

HD

No. of animals per sex per dose:

25 females/dose group

Control animals:

yes, concurrent vehicle

Parental animals: Observations and 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 2
0.
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, t
remors, 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 par
turition or bizarre behaviour were recorded.

Postmortem examinations (offspring):

Post-Mortem Examination
On gestation day 20, sperm positive (presumed pregnant females) were subjected to a caesarean se
ction 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 f
ormaldehyde.
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 pe
r animal) for serum levels of thyroid hormones and pituitary-thyroid axis hormones (T3, T4, TSH). Ho
rmone 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 sac
rificed at the end of the treatment period.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, sl
ides, 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.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Increased salivation was noted in 1/25 females of the MD group and 3/25 females of the HD. Moving
the bedding was observed in 6/25 females of the MD group, and 25/25 females of the HD group on
several days of treatment. Moving the bedding and salivation were seen transiently and considered
as slight clinical signs elicited by local effects of the test item formulation and/or attributed to discomfo
rt of the animals due to the oral administration, but not systemic toxicity.
Slight clinical signs like hairless areas in 1/25 females of the LD group and 2/25 females of the MD gr
oup, a scratch/cut in 1/25 females of the control group and piloerection in 1/25 females of the MD g
roup were noted in single animals without dose-dependency and were not considered toxicologically
relevant.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid Hormones
No test item-related effect of toxicological relevance was observed on maternal thyroid hormone (T3, T4, TSH) levels in the test item-treated groups when compared to the controls.
Mean T4 values were observed to be statistically significantly higher in the LD (12% above control)
and MD (11% above controls) group when compared to the control group. However, as this finding
showed no dose-dependency and mean T3 value of the HD group remained unaffected it was not
considered test-item related. Moreover, values were within the range of historical control data.
Mean T3 and TSH values were comparable between test item-treated groups and the control group.

Description (incidence and severity):

Histotechnique was performed on thyroid gland samples from all females from the main study.
Under the conditions of this study, the test item did not induce morphological lesions in the thyroid gland. In correlation with no statistically significant differences in thyroid/parathyroid weight and macroscopic observation, histopathological evaluation revealed no test item-related effects.

Histopathological findings: non-neoplastic:

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

not examined

Mortality / viability:

no mortality 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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean foetal weight on a per litter basis showed no toxicologically relevant effects in the test itemtreated groups when compared to the control group.
Mean foetal weight on a foetal basis revealed statistically significant higher mean male (3% above
controls) and female (4% above controls) foetus weight in the MD group and statistically significan tly higher mean male (3% above controls) foetus weight in the HD group when compared to the corresponding control group. However, due to the slightness of this effect and the lack of dose-dependency it was not considered toxicologically relevant.

Conclusions:

On the basis of this prenatal developmental toxicity study (according to OECD 414, 2018) 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 bodyweight/day.

Reference 3

Endpoint:

reproductive toxicity, other

Remarks:

other: 90d gavage test in rats including fertility parameters and hormone analysis (T3, T4 and TSH)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-12-03 until 2019-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 408

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

No. of animals per sex per dose:

10

Clinical signs:

effects observed, non-treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

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.

Endocrine findings:

no effects observed

Description (incidence and severity):

Statistical significance was found in the male LD group for a increase of 80 % compared to control for T3 mean hormone level. The statistically significant change from control group was found without dose dependency and therefore the single finding is not considered to be of toxicological relevance.
No statistical significances were noted for mean hormone levels in females when compared to control group.

Organ weight findings including organ / body weight ratios:

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

Reproductive function: oestrous cycle:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effect on fertility parameters in this 90-day oral toxicity study

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.

Conclusions:

In this sub-chronic study, the safety of MPAAU was examined in Wistar rats (four groups of 10 males and 10 females each). The test substance was administered as a solution in tap water by daily oral gavage during 90 consecutive days, at levels of 0 (tap water only), 100, 300 or 1000 mg MPAAU/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.
Especially, there were no effects of the test substance on organ weight, macroscopic examination or histopathology of organs and tissues of the reproductive system (ovaries, oviducts, testes, prostate and seminal vesicles with coagulating glands as a whole, uterus with cervix and vagina). Further, no test item-related adverse effect on thyroid hormone levels (T3, T4, TSH) has been observed.
The no observed adverse effect level (NOAEL) of the test item MPAAU in this sub-chronic study is considered to be 1000 mg/kg body weight/day.

Reference 4

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 for cross-reference:

reference to other study

Qualifier:

according to guideline

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. QA statement)

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

Critical effects observed:

no

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:

Supportiung GLP studies (OECD 407, 408, 414 (two species)) with Klimisch score 1 available, but no mating/no data in offspring available (=screening level).
Justification for non-submission of data from an reproductive toxicity study OECD 443 / 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.

A 90-Day Repeated Dose Oral Toxicity study with the test item MPAAU in male and female Wistar rats, with dose levels of 100, 300 and 1000 mg/kg body weight/day evidenced the following conclusions:
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.
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.

A prenatal developmental toxicity study (according to OECD 414, 2018) 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 has been conducted. 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 (including hormone analysis) 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.

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 and some retatdartions in ossification were observed. These effects are deemed to be secondary to the marked maternal toxicity. The NOAEL (maternal & embryotoxicty) was placed at the mid-dose level, 300 mg/kg bw/day.

 

Short description of key information:
OECD 407 including fertility parameters (rat): NOAEL (fertility) = 1000 mg/kg bw/day (limit dose).

OECD 408 including hormome analysis (rat): NOAEL (fertility) = 1000 mg/kg bw/day (limit dose).

OECD 414 incliding hormone analysis (rat): NOAEL (maternal + embryotoxicity) = 1000 mg/kg bw/day (limit dose).

OECD 414 (rabbit): NOAEL (maternal & 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)

Evidence for no 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, testicular spermcount and daily sperm production) and hormone analysis (T3, T4, TSH) in rats of a repeated-dose toxicity test up to and including the limit dose.

Effects on developmental toxicity

Description of key information

OECD 414 (rabbit): negative.

OECD 414 (rat): negative.

Link to relevant study records

Reference

Reference 1

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Conditional decline: 10 rabbits in the HD group showed blood aroud the perineum and/or in the cage.

Mortality:

mortality observed, treatment-related

Description (incidence):

One rabbit of the HD group was found dead on GD 9. Two other rabbits of this group were killed in extremis on GD 13 and GD 21.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weight loss or growth retardation, and reduced feed intake were noted in the HD group.
These changes were evident in the initial phase of the study , but remained after reduction of te HD level to 400 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatrment related macroscopic findings of the dams were observed at Caesarean section. The carcass weight was reduced in the HD group.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Two HD rabbits had an early delivery and were necropied (during partus) on GD 28.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

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.

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

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal weights were decreased in the mid- and high-dose group.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

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 may be
secondary to reduced maternal feed intake in these groups.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

reduction in number of live offspring

other: embryotoxicity

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

other:

Description (incidence and severity):

No visceral or skeletal malformations or anomalies were observed at any dose level that are treatment related.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

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 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.

Reference 2

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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):

Increased salivation was noted in 1/25 females of the MD group and 3/25 females of the HD. Moving the bedding was observed in 6/25 females of the MD group, and 25/25 females of the HD group on several days of treatment. Moving the bedding and salivation were seen transiently and 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.
Slight clinical signs like hairless areas in 1/25 females of the LD group and 2/25 females of the MD group, a scratch/cut in 1/25 females of the control group and piloerection in 1/25 females of the MD group were noted in single animals without dose-dependency and were not considered toxicologically relevant.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in any of the groups in the course of this study. All animals of the test item-treated groups and the control group survived the scheduled study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight increased in all groups before initiation of treatment and further increased with the progress of the study comparably in all test item-treated groups and the controls.
There were no biologically relevant or statistically significant differences were observed in body weight development when comparing test item-treated groups with the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by the treatment with the test item.
Test item-treated groups showed no statistically significant or biologically relevant differences in food consumption compared to the controls.

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:

no effects observed

Description (incidence and severity):

Thyroid Hormones
No test item-related effect of toxicological relevance was observed on maternal thyroid hormone (T3, T4, TSH) levels in the test item-treated groups when compared to the controls.
Mean T4 values were observed to be statistically significantly higher in the LD (12% above control) and MD (11% above controls) group when compared to the control group. However, as this finding showed no dose-dependency and mean T3 value of the HD group remained unaffected it was not considered test-item related. Moreover, values were within the range of historical control data.
Mean T3 and TSH values were comparable between test item-treated groups and the control group.

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 when comparing test item-treated groups and the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Thyroid/parathyroid weight showed no statistically significant or biologically relevant effects when comparing dosing groups to the control group.
No test item-related necropsy findings were observed up to the dose of 1000 mg/kg bw/day.
The single finding of few kidney cysts in 1/25 females of the MD group (female no. 71) was not considered test item-related as no other animals of any of the dose groups showed similar findings.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In a study phase (AnaPath Phase Number 12549 C), Histotechnique was performed on thyroid gland samples from all females from the main study (Eurofins Munich / BSL Munich Study No.: 186030) which was performed at BSL Bioservice Scientific
Laboratories Munich GmbH, Planegg / Germany.
Under the conditions of this study, the test item did not induce morphological lesions in the thyroid gland. In correlation with no statistically significant differences in thyroid/parathyroid weight and macroscopic observation, histopathological evaluation
revealed no test item-related effects.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

Successful mating resulted in 24/25 pregnancies in the LD group, 24/25 pregnancies in the MD group and 24/25 pregnancies in the HD group when compared to 23/25 pregnancies in the control group.
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
No biologically relevant or statistically significant differences were observed in mean maternal terminal body weight, gravid uterus weight and resulting adjusted maternal weight when comparing test item-treated groups and the control group.

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

Description (incidence and severity):

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses 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

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean foetal weight on a per litter basis showed no toxicologically relevant effects in the test item-treated groups when compared to the control group.
Mean foetal weight on a foetal basis revealed statistically significant higher mean male (3% above controls) and female (4% above controls) foetus weight in the MD group and statistically significantly higher mean male (3% above controls) foetus weight in the HD group when compared to the corresponding control group. However, due to the slightness of this effect and the lack of dose-dependency it was not considered toxicologically relevant.

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):

There were no external abnormalities considered to be of toxicological relevance in any of the test item-treated groups. Statistical analysis of foetal and litter incidences showed no significant differences compared to the control group.
One foetus of the MD group was observed with absent lower jaw, mouth, tongue and a misshapen head and another foetus of the MD group showed the findings of an absent lower jaw and an open eye. However, without the observation of similar findings in any other foetuses of the dose groups these findings were not considered toxicologically relevant.
The findings of head (neck) haematoma in one foetus of the control group and one foetus of the MD group showed no dose-dependency and thus were not considered test item-related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no general trend towards dose-dependently altered ossification. As usual for foetuses at this stage of gestation (day 20) incomplete ossification was seen in several bones of several litters in all groups including control. Bones of the skull, sternum, paws, pelvis and vertebra were affected by slight variations in the status of expected ossification in terms of incomplete ossification, misaligned ossification, irregular ossification, unossification, supernumerary ossification, bipartite ossification, dumbbell ossification or increased ossification throughout all groups. Values showed no dosedependent pattern and litter incidences for ossification status were statistically insignificant between the dose groups and controls except for the finding of incomplete ossification of the squamosal bone (0 % in the HD group compared to 45 % in controls) which was not considered of relevance as the control group showed a higher incidence
than the test item-treated HD group.
Statistically significant higher foetal incidence of incomplete ossification of caudal arches was observed in the LD group compared to the control group (54% in the LD group compared to 22% in the HD group). However, the MD and HD group showed no similar observation and thus higher incidence in the LD group was assumed to be incidental. Statistically significantly higher foetal incidence of unossified caudal arches was observed in the LD and MD group compared to the controls (96% in the LD group and 92% in the MD group compared to 47% in the control group). As this observation followed no dose-dependency and the HD group showed no statistically significant differences it was not considered toxicologically relevant.
Scapulae were observed with findings in several foetuses from all dose groups. Misshapen scapulae were observed with in one single foetus of the HD group. Bent scapulae showed a litter incidence of 10% in the control group, 5% in the LD group, 15% in the MD group and 0% in the HD group. Short scapulae were observed with a litter incidence of 10% in the control group, 5% in the LD group, 15% in the MD group and 10% in the HD group. However, due to the low incidences of altered scapulae and the lack of statistical significance or dose dependency they were not considered toxicologically relevant.
A dose dependent trend was observed in the litter incidence of bipartite (5% in the control group, 5% in the LD group, 10% in the MD group and 15% in the HD group) and irregular (15% in the control group, 20% in the LD group, 35% in the MD group and 50% in the HD group) ossification of thoracic centra. However, due to the low number of affected foetuses in the test item-treated groups and the occurrence of these findings also in foetuses of the control group this was considered as variation.
Several findings (misaligned ossification, split, fused) were observed during examination of sternebra. However, these findings showed no statistical significance or dosedependency and thus were not considered test item-related.
Supernumerary (14th) thoracolumbal ribs were noted in several foetuses throughout all groups including the control group. The litter incidence of supernumerary thoracolumbal ribs (bilateral/right side/left side and rudimentary/full) showed no statistically significant differences or dose-dependent trend when comparing test item-treated groups with the control group. Foetal incidence of rudimentary ribs (bilateral) was observed to be statistically significantly higher in the HD group (42%) and showed a dose-dependent trend in LD (27%) and MD (29%) group compared to the control group (19%). Rudimentary cervical ribs (left side or right side) were noted in few foetuses throughout all groups including control without statistically significant difference. Cervical ribs are skeletal alterations that can occasionally be seen in animals of this strain and age. Rudimentary/short ribs are considered as transient abnormalities [14][15] and not considered adverse.
The finding of wavy ribs was observed in all groups without statistical significance or dose dependency (litter incidence C: 45%, LD: 30%, MD: 60%, HD: 20%) and thus was not considered test item-related. In general, wavy ribs are common findings in rodent studies and are classified as variations which are considered to be postnatally reversible [12][13].
Pelvic girdle caudal shift (bilateral / left side / right side) was noted in few foetuses throughout all groups including control without statistically significant difference to controls and without a dose-dependent trend. Changes of the position of the pelvic girdle relative to the number of pre-pelvic vertebrae can occasionally be seen in animals of this strain and are not considered test item related.
Few findings (misshapen mandibles and tympanic annuli in one foetus of the MD group, fused parietal and frontal bone in one foetus of the control group and 2 foetuses of the MD group, fused zygomatic bone and process (of maxilla) in 3 foetuses of the MD and two foetuses of the HD group, fused sacral centra and arches in 2 foetuses of the control group, 3 foetuses of the LD group and one foetus of the HD group and fused caudal centra and arches in one foetus of the control group, 2 foetuses of the LD and MD group, each, and one foetus of the HD group) were observed occasionally 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 foetal viscera by free hand microdissection technique revealed a range of findings in all test item-treated 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.
Affected organs/tissues were the umbilical artery (malpositioned, in 8 foetuses of the control group, 11 foetuses of the LD group, 12 foetuses of the MD group, 10 foetuses of the HD group), the stomach (discoloured, in one foetus of the MD group), the liver (supernumerary lobe, 5 foetuses of the control group, 3 foetuses of the LD group, 6 foetuses of the MD group, 3 foetuses of the HD group), the pancreas (discoloured, 5 foetuses of the control group, 12 foetuses in the LD group, 13 foetuses in the MD group, 13 foetuses of the HD group), the testis (malpositioned, 3 foetuses of the control group, 4 foetuses of the LD group, 7 foetuses of the MD group, 3 foetuses of the HD group), the azygos vein (bilateral, 4 foetuses in the control group, one foetus in the LD group, 3 foetuses in the MD group, 8 foetuses of the HD group; malpositioned, one foetus of the control group), the thyroid gland (large, one foetus of the control group), the
lung lobe (small, one foetus of the control group, the kidneys (discoloured, one foetus of the LD group and one foetus of the MD group; dilated renal pelvis, one foetus in the LD group, 4 foetuses of the MD group, 3 foetuses of the HD group), the spleen (discoloured, one foetus of the control group; small, one foetus of the LD group) and the thymus (long, 9 foetuses of the control group, 15 foetuses of the LD group, 12 foetuses of the MD group, 8 foetuses of the HD group). Abdominal haemorrhages were observed in 11 foetuses of the control group, 13 foetuses of the LD group, 16 foetuses of the MD group and 18 foetuses of the HD group and subcutaneous haemorrhages in the neck occurred in 18 foetuses of the control group, and 20 foetuses in each dose group (LD,
MD, HD).
The incidence of listed findings in the test item-treated groups was generally at frequencies comparable to the controls or in some cases slightly higher or lower in test item-treated groups compared to controls. Thus, these findings were not considered test item-related.
Statistically significantly higher mean foetal incidence (on a litter basis) of a discoloured pancreas in the MD group (31%) compared to the control group (8%) was not considered toxicologically relevant as no dose-dependency was observed for this finding.
Testicular descent was observed to be complete in all examined male foetuses. No effect of the test item on testicular descent was evident in test item-treated male foetuses when compared to male control foetuses.

Other effects:

no effects observed

Description (incidence and severity):

Craniofacial Examination
There were no craniofacial abnormalities of toxicological relevance in any of the test item-treated groups. Statistical analysis of foetal and litter incidences showed no significant differences compared to the control group.
Single findings including subcutaneous haematoma (head) and a misshapen cerebrum in one foetus of the HD group, subcutaneous haematoma (head) in one fetus of the MD group, large cerebrum in one foetus of the MD group, dilated ventricles (lateral and 3rd ventricle in one foetus of the control group), perimeningal space filled with red material in 2 foetuses of the control, 2 foetuses of the LD and 3 foetuses of the MD group, an increased perimeningal space in 3 foetuses of the control group, one foetus of the LD group, one foetus of the MD group and 2 foetuses of the HD group were observed without dose-dependency and were not related to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effect found.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Conclusions:

On the basis of this prenatal developmental toxicity study (according to OECD 414, 2018) 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.