Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

MPAAU is not a reproductive toxicant.

Link to relevant study records
Reference
Endpoint:
reproductive toxicity, other
Remarks:
other: 28d gavage test in rats including fertility parameters
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-28 till 2011-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD 407 (Oct 2008), including fertility parameters
Deviations:
yes
Remarks:
The rats were about 8-9 weeks old at the start of the treatment period (rationale: to enable evaluation of the oestrus cycle during the last two weeks of the study).
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The rats, 20 males and 20 females, were about 8-9 weeks old at the start of the treatment period (rationale: to enable evaluation of the oestrus cycle during the last two weeks of the study). The body weights at initiation of treatment were within ± 20% of the mean weight for each sex, and ranged from 235-286 g (mean 259 g) for males and from 165-196 g (mean 179 g) for females.
Details on mating procedure:
no mating
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content, homogeneity and stability of the test substance in the carrier (tap water) were confirmed by HPLC-UV analysis.
Samples were taken from each dosing formulations prepared in the study. Analyses to determine the content, homogeneity and stability of the test substance in the carrier were conducted by HPLC-UV analysis (after storage in the animal room for approximately four hours and after storage in the refrigerator (2-10oC) for 7 days)
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
50, 300, 1000 mg MPAAU/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
General clinical observations
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

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

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

Feed consumption
Feed consumption was measured per cage by weighing the feeders. The consumption was measured over successive periods of 3 or 4 days throughout the treatment period. The results were expressed in g per animal per day.



Haematology
Haematology was conducted on all surviving animals. At necropsy, blood samples were taken from the abdominal aorta of the rats whilst under CO2/O2 anaesthesia. The rats were fasted overnight before necropsy (water was freely available). EDTA was used as anticoagulant. In each sample the following determinations were carried: haemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), reticulocytes, total white blood cells (WBC), differential white blood cells, prothrombin time, thrombocytes.
The following parameters will be calculated:
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

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

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

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

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

Histopathological examination
The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs listed above (except those marked with an asterisk) of all animals of the control group and the high-dose group, Gross lesions were examined in rats of all dose groups.
Oestrous cyclicity (parental animals):
Vaginal smears to evaluate the oestrus cycle length and normality were made daily from day 15 until sacrifice on day 28. Smears were made and stained of all females, but evaluated only in the control and high-dose group.
Sperm parameters (parental animals):
Epididymal sperm motility, count and morphology
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups, and the motility was measured. The cauda epididymidis was dissected, weighed and thereafter minced in 3 ml M199 medium containing 0.5% BSA. Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups with the Hamilton Thorne Integrated Visual Optical System (IVOS).
In addition, a smear of the sperm solution was prepared and stained for all males, and two hundred spermatozoa of the smear of each male of the control group and high-dose group were analysed.
Litter observations:
no mating/offspring
Postmortem examinations (parental animals):
Testicular sperm count
At scheduled necropsy, the left testis of all males of all groups was placed on dry ice and subsequently stored in a freezer (<-70°) for later determination of the number of homogenization-resistant spermatids. Before analysis the testis was thawed and the tunica albuginea removed. After weighing, testicular parenchyma was minced and homogenized in 25 ml Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS in males of the control group and high-dose group. The daily sperm production was calculated as ‘number of spermatozoa per gram testicular parenchyma / 6.1’ (WF Blazak et al.; in Male Reproductive Toxicology, eds. RE Chapin and JJ Heindel, Methods in toxicology volume 3A, 1993).
Postmortem examinations (offspring):
no mating/offspring
Statistics:
Numerous tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Because numerous variables are subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results is based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings.
Reproductive indices:
no mating/offspring
Offspring viability indices:
no mating/offspring
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not specified
Fertility parameters
Estrus cyclicity: Smears obtained during the last 14 days prior to sacrifice of all control females and high-dose females were evaluated. The number of acyclic females, the mean length of the longest cycle, the number of cycles per animal and the number of females with a prolonged estrus period were comparable in both groups.

Sperm analysis
Epididymal sperm motility: Statistical analysis of sperm motility data indicated no significant differences between the test groups and the controls.
Epididymal sperm count: No statistically significant effects on epididymal sperm counts were observed.
Epididymal sperm morphology: No statistically significant effect on sperm morphology was observed.
Testicular sperm count: No effects on the number of spermatozoa per gram testicular parenchyma or on daily sperm production were observed.
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
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
Conclusions:
The no-observed-adverse-effect level (NOAEL) was set at the highest dose level, 1000 mg MPAAU/kg bw/day.
Executive summary:

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 (estrus cyclicity, 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 set at the highest dose level, 1000 mg MPAAU/kg bw/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:
High quality GLP study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The 28-day study in rats covers the critical observations which would be needed to assess the effects of a substance on fertility. According the REACH guidance document on information requirements and chemical safety, no further studies are required for to assess fertility if the 28-day study indicates adverse effects on reproductive organs or tissues.


There were no effects of the test substance on fertility parameters (oestrus cyclicity, epididymal sperm motility, sperm count and sperm morphology, and testicular sperm count 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.

Effects on developmental toxicity

Description of key information
MPAAU is not a developmental toxicant.
Link to relevant study records
Reference
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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Ninety time-mated female New Zealand White rabbits of 4-5 months were obtained from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated females arrived at the testing facilities on GD 1 (23-25 November 2011 for Lot 1, 2 and 3, respectively). The rabbits were checked for overt signs of ill health and abnormalities upon arrival.
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content, homogeneity and stability of the test substance in the carrier (tap water) were confirmed by HPLC-UV analysis.
Dilutions of the test substance in the vehicle were prepared weekly (namely on 25 November, 2 December, 5 December (only the adapted high-dose level), 9 December and 16 December 2011), and used within 9 days after preparation. The dilutions were stored in a refrigerator (2-10°C) in portions sufficient for one day. The vehicle for dosing the controls was similarly stored.
Details on mating procedure:
Ninety time-mated female New Zealand White rabbits of 4-5 months were obtained from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated females arrived at the testing facilities on GD 1 (23-25 November 2011 for Lot 1, 2 and 3, respectively).
Duration of treatment / exposure:
22 consecutive days
The test substance was administered once daily by oral gavage, as a dilution in tap water from GD 6 up to and including GD 28. Controls were treated with vehicle (tap water) only. The animals were dosed for the last time on the day prior to necropsy.
Frequency of treatment:
once daily
Duration of test:
Arrival of time-mated animals : 23-25 November 20111 (on GD 1)2
Start of the treatment : 28-30 November 20111 (from GD 6)
Date of scheduled necropsy : 21-23 December 20111 (GD 29)
Remarks:
Doses / Concentrations:
0, 50, 300, 500/400 mg MPAAU/kg bw/day
Basis:
analytical conc.
No. of animals per sex per dose:
22 pregnant females/group
Details on study design:
On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14.
Maternal examinations:
Clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
Animals showing signs of severe intoxication, particularly if death appeared imminent, were humanely killed to prevent loss of tissues by autolytic degeneration.

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

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

Water consumption: Water consumption was not measured.

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

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


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

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

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

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

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

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

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

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

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

Visceral examination

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

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

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

Skeletal examination

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

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

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

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

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

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

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.

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

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

No frank malformation were observed. The visceral variation in the high dose group, and the skeletal retardations in ossification in high and mid dose groups are considered transient in nature and deemed to be secondary to reduced maternal feed intake and the overall growth retardation in these groups. Therefore, the results indicate that MPAAU was not teratogentic in the NZW rabbit. The developmental no-observed-adverse-effect level (NOAEL) was 500 mg MPAAU/kg body weight/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The test substance on its on has not been tested so results and endpoint selection should take this into account. A developmental screening study was conducted in New Zealand White rabbits and high doses were used in order to set appropriate dose groups for the main study. The results are consistent and can be used set a NOAEL for this endpoint.
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

An OECD 414 guideline study was conducted on MPAAU. The selected doses were 0, 50, 300, 500/400 mg MPAAU/kg bw/day.

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 post-implantation 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. retardations in ossification, the total incidence of fetuses showing skeletal retardations was increased in the mid- and 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 pericardium and abdomen (high-dose group). No other statistically significant visceral variations were observed.

Based on the above data on developmental toxicity, it can be concluded that MPAAU is not reproductive toxicant and should not be classified.

NOAEL maternal toxicity for MPAAU is set at 300 mg/kg bw/day

NOAEL teratogenicity for MPAAU is set at 400 mg/kg bw/day

NOAEL embryotoxicity for MPAAU is set 300 mg/kg bw/day

Justification for classification or non-classification

No classification applicable for toxicity for reproduction.