Methylphosphonic acid, compound with amidinourea (1:1)

Endpoint summary

Description of key information

RD oral: NOAEL (rat) = 1000 mg/kg bw/day (OECD 407); no hazard identified; RC not required

RD oral: NOAEL (rat) = 1000 mg/kg bw/day (OECD 408) 25 June 2018; no hazard identified; no DNEL necessary

RD dermal: waived (scientifically not justified), WoE: no classification necessary; no DNEL necessary
RD inhalation: waived (exposure considerations), WoE: no classification necessary; no DNEL necessary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-28 till 2011-11-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP and without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 3 October 2008

Deviations:

yes

Remarks:

The rats were about 8-9 weeks old at the start of treatment in order to enable evaluation of the oestrus cycle (fertility parameters) during the last two weeks of the study. The deviations were considered not to have affected the validity of this study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats, 20 males and 20 females, were about 8-9 weeks old at the start of the treatment period (rationale: to enable evaluation of the oestrus cycle during the last two weeks of the study). The body weights at initiation of treatment were within ± 20% of the mean weight for each sex, and ranged from 235-286 g (mean 259 g) for males and from 165-196 g (mean 179 g) for females.

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content, homogeneity and stability of the test substance in the carrier (tap water) were confirmed by HPLC-UV analysis.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
50, 300, 1000 mg MPAAU/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

5 males and 5 females per group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

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

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

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

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

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

Sacrifice and pathology:

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

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

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

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

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

Histopathological examination
The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs listed above (except those marked with an asterisk) of all animals of the control group and the high-dose group, Gross lesions were examined in rats of all dose groups.

Other examinations:

Fertility parameters
Oestrus cyclicity
Vaginal smears to evaluate the oestrus cycle length and normality were made daily from day 15 until sacrifice on day 28. Smears were made and stained of all females, but evaluated only in the control and high-dose group.

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

Testicular sperm count
At scheduled necropsy, the left testis of all males of all groups was placed on dry ice and subsequently stored in a freezer (<-70°) for later determination of the number of homogenization-resistant spermatids. Before analysis the testis was thawed and the tunica albuginea removed. After weighing, testicular parenchyma was minced and homogenized in 25 ml Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS in males of the control group and high-dose group. The daily sperm production was calculated as ‘number of spermatozoa per gram testicular parenchyma / 6.1’ (WF Blazak et al.; in Male Reproductive Toxicology, eds. RE Chapin and JJ Heindel, Methods in toxicology volume 3A, 1993).

Statistics:

Numerous tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Because numerous variables are subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results is based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

Fertility parameters
Estrus cyclicity: Smears obtained during the last 14 days prior to sacrifice of all control females and high-dose females were evaluated. The number of acyclic females, the mean length of the longest cycle, the number of cycles per animal and the number of females with a prolonged estrus period were comparable in both groups.

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

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:

not specified

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 (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 placed at the highest dose level, 1000 mg MPAAU/kg body weight/day.

Executive summary:

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

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

or in macroscopic- and microscopic findings.

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

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

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-03 until 2019-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 consecutive days

Dose / conc.:

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

Remarks:

measured concentrations

Dose / conc.:

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

Remarks:

measured concentrations

Dose / conc.:

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

Remarks:

measured concentrations

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Food consumption was measured weekly during the treatment period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. Inadvertently, clinical observations were not made on one day (25 February 2019, study day 74, 75, 76 or 77 respectively (staggered start)).
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

Functional Observations
Once before the first exposure and once in the last week multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests [14]. These tests were conducted in all animals.

Sacrifice and pathology:

Haematology
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined:
haematocrit value (HCT)
haemoglobin content (HGB)
red blood cell count (RBC)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin (MCH)
mean corpuscular haemoglobin concentration (MCHC)
reticulocytes (Ret)
platelet count (PLT)
white blood cells (WBC)
neutrophils (Neut)
lymphocytes (Lym)
monocytes (Mono)
eosinophils (Eos)
basophils (Baso)
large unstained cells (Luc)

Blood Coagulation
Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined:
prothrombin time (PT)
activated partial thromboplastin time (aPTT)

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined:
alanine aminotransferase (ALAT)
aspartate-aminotransferase (ASAT)
alkaline phosphatase (AP)
creatinine (Crea)
total protein (TP)
albumin (Alb)
urea
total bilirubin (TBIL)
total bile acids (TBA)
total cholesterol (Chol)
low density lipoprotein (LDL)
high density lipoprotein (HDL)
triglycerides (TG)
glucose (Gluc)
sodium (Na)
potassium (K)
For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and stored at < -15 °C. T3, T4 and TSH serum levels were determined of main study animals (80 animals).

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour / appearance were recorded. The following parameters (Table 7) were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).
specific gravity
nitrite
pH-value (pH)
protein
glucose
ketone bodies
urobilinogen (UBG)
bilirubin (BIL)
erythroctes (Ery)
leukocytes (Leuc)

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

Organ Weight
The wet weight of the organs (Table 8) was taken from sacrificed animals as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of the animal euthanised on study day 8 for animal welfare reasons were not recorded:
liver, uterus with cervix
kidneys, thymus
adrenals, thyroid / parathyroid glands (were weighed after fixation)
testes, spleen
epididymides, brain
prostate, seminal vesicles and coagulating glands, pituitary gland
ovaries, heart
The following tissues from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.
adrenal glands x x
all gross lesions x x
aorta x x
brain (incl. medulla/pons, cerebellar and cerebral
cortex) x x
caecum x x
colon x x
duodenum x x
epididymides x x
eyes with optic nerve and Harderian gland x --
femur with knee joint x --
heart x x
ileum (including Peyer´s patches) x x
jejunum x x
kidneys x x
liver x x
lungs x x
lymph nodes (mandibular) x --
lymph nodes (mesenteric and axillary) x x
mammary gland area (male and female) x x
oesophagus x x
ovaries x x
oviducts x --
pancreas x x
pituitary x x
prostate and seminal vesicles with coagulating glands as a whole x x
rectum x x
salivary glands (sublingual, submandibular, parotis) x x
sciatic nerve x x
skeletal muscle x x
skin x x
spinal cord (cervical, thoracic and lumbar segments) x x
spleen x x
sternum (with bone marrow) x x
stomach x x
testes x x
thymus x x
thyroid gland including parathyroid glands x x
tongue x --
trachea x x
ureters x --
urinary bladder x x
uterus with cervix and vagina x x
The animal intercurrently euthanised for animal welfare reasons was also subjected to a gross necropsy and the organs preserved for a histopathological examination.

Histopathology
The afore-listed organs (Table 9) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical findings for male control animal no. 4, which was euthanized on study day 8, were prone position, severely reduced spontaneous activity, eyes closed and abnormal breathing. No clinical findings were seen on treatment days 1 to 7. The cause of morbidity was not evident at the histopathological evaluation and is assumed to be related to gavage procedure.
In the female HD group, prone position was noted on treatment days 68 to 73 for animal no. 77 and dehydration on day 15 for HD group female animal nos. 71 to 75. As these findings were observed in one single animal or on one single day, a toxicological effect of the test item is not considered.
The findings crust and/or hairless area at the cheek, head or forelimbs in several female animals of the control group (animals no. 41, 47, 49 and 50) or for female LD group animal no. 57 (hairless area at forelimbs, hindlimbs, lateral area, abdomen, legs) are not considered to be of biological or toxicological relevance.
Moving the bedding and/or slight to moderate salivation (in one HD female animal with a severe grade) were noted in 4 out of 10 male LD group animals and in all male and female MD and HD group animals. The clinical signs salivation and moving the bedding were observed in close timely relation to the dose administration and therefore considered to be signs of a local reaction after test item treatment rather than a systemic adverse effect.
Statistical significance was noted for males in week 3 for a slightly increased mean score of salivation in the male HD group (15 % above control), in week 12 for animal sleeps (87.1 % below control) and animal moving in cage (305 % above control) in the male MD group.
In females, statistical significance was found in week 2 for an increase of 10 % above control for response to handling in the LD group, for an increase of salivation in the HD group in week 3 (20 % above control), in week 4 (30 % above control), in week 5 (25 % above control), in week 6 (25 % above control), in week 8 (20 % above control), in week 10 (35 % above control) and additionally in week 11 (30 % above control) and in the HD group for an increased mean score of animal moving in cage in week 6 (100 % above control).
The statistically significant effects on clinical parameters were not considered to be test item-related as they were seen occasionally in single weeks. Statistically significantly increased salivation was observed in several weeks in the female HD group but not in the male HD group, where the statistical significant increase was seen in one single week during the entire treatment period. A systemic adverse effect of the test item is therefore not considered.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the male control group, one animal (animal no. 4) was euthanized in moribund condition on study day 8. Clinical findings on day of sacrifice were prone position, severely reduced spontaneous activity, eyes closed and abnormal breathing. No clinical findings were seen on treatment days 1 to 7. Considering histopathological assessment, death of animal no. 4 was considered to be incidental. The cause of morbidity was not evident at the histopathological evaluation and is assumed to be related to gavage procedure.
There were no mortalities in the remaining males and females of the control and all dose groups until the end of the treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related effect on body weight development was found during the entire treatment period.
There were no statistical significances in weekly mean body weight or body weight change. The mean values of all male and female dose groups were comparable to the respective control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no test item-related effect on food consumption in any of the dose groups.
Mean daily food consumption of all male and female dose group animals was comparable to corresponding control groups.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were found for all haematological and coagulation parameters of all male and female dose groups at the end of the treatment period.
A statistical significant decrease of 71.4 % below control was found for the mean value of eosinophils in the male MD dose group and for an increase of monocytes in the female MD dose group (51.1 % above control). The statistical significant changes from control group were found without dose dependency and therefore the single findings are not considered to be of toxicological relevance.
No statistical significances were noted for coagulation parameters in any male and female dose group when compared to control group.
Overall, all other mean values for all parameters of haematology and coagulation in the LD, MD and HD dose groups were comparable to corresponding control groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test item had no toxicologically relevant effects on parameters of clinical biochemistry or hormone analysis of males and females in any of the test item-treated groups.
Statistical significance was found in the female HD group for a decrease of 64.87 % below control for total bile acid. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency. Additionally, no test item- related effects were noted at histopathological assessment. Therefore, the statistical significance is considered to be of no biological or toxicological relevance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no adverse effect of the test item on urinary parameters measured at the end of the treatment period.
Elevated levels of bilirubin in single animals of the male HD group, protein in several animals of all male dose groups, glucose in one male MD animal, erythrocytes in one male HD and female MD animal and leucocytes in several animals of all male dose
groups when compared to control, were not considered to be of toxicological relevance as no adverse effects were seen in parameters of clinical biochemistry and histopathological evaluation.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment for the male and female groups.
The statistical significant mean scores (decrease of animal sleeps, increase of moving in cage) for the male LD group in the last week of treatment were not considered to be an effect of test item treatment as they were seen without dose dependency.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment with the test item had no toxicologically relevant effects on absolute and relative organ weight of males and females in any of the test item-treated groups.
No statistical significant organ weight changes were found for all male and female dose groups when compared to control group. Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic finding for the animal sacrificed in moribund condition (control animal no. 4) was a dark red coloured right caudal lobe of the lung. Considering histopathological assessment, the cause of morbidity was not evident at the histopathological evaluation.
In HD male animal no. 31 a cyst at the prostate gland and spotted harderian glands were observed at necropsy on study day 91. Regarding the female HD group, the stomach of animal no. 74 was seen with few black foci, the axillary lymph node of animal no. 77 and the mandibular lymph nodes of animal no. 73 were red coloured. A dilatation of the uterus (completely/both sides) was found for control animal no. 50, MD animal no. 70 and HD animals no. 71, 73, 74 and 80.
Considering histopathological evaluation, all gross lesions observed at necropsy for terminally sacrificed animals were deemed to be incidental or represented the normal animal physiology and therefore, considered to be not related to the treatment with the test item.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed during the histopathological evaluation. All findings recorded in decedent and surviving animals were deemed to be incidental or were within the range of background alterations that may be recorded in animals of this strain and age and in this study type.
Considering histopathological assessment, there were no organ weight changes, gross lesions or histological alterations that could be attributed to the treatment with the test item. Thus, the histopathological NOEL (no observed effect level) could be established at 1000 mg/kg bw.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: no adverse effect observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

On the basis of the present study, the 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 the following conclusions can be made:
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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study with Klimisch score 1 available.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Repeated dose toxicity, oral:

One fully reliable study in rats is available. Based upon the absence of any adervese effect in this study, the "no-observed-adverse-effect-level" of the substance is 1000 mg/kg body weight for male and female rats.

- Repeated dose toxicity, dermal:

No study available. According to Section 1 of Annex XI to Regulation EC NO 1907/2006 testing is not scientifically necessary based on sufficient weight of evidence. The repeated oral dose toxicity of the substance has been assessed without any adverse effect (see IUCLID Chapter 7.5.1). The toxicity potential can be extrapolated to dermal route of administration. The dermal absorption of the active ingredient form technical substance has been investigated (see IUCLID Chapter 7.1.2). The low penetration rate (5%) makes it unlikely that the substance could be hazardous when exposed dermally.

- Repeated dose toxicity, inhalation:

No study available. According to Section 3 of Annex XI to Regulation EC NO 1907/2006 testing is not scientifically necessary. The substance is unlikely to be inhaled for the following reasons:

The substance is an organic salt and has a very low vapour pressure and a high melting point. The potential for the generation of inhalable forms is considered to be low. The use of this substance will not result in aerosols, particles or droplets of an inhalable size, so the exposure to humans via inhalation will be unlikely to occur.

In accordance with Section 1 of REACH Annex XI, the study does not need to be conducted based on sufficient weight of evidence (WoE):

In view of the oral NOAEL >1000 mg/kg bw/day (see IUCLID Chapter 7.5.1) and assuming equivalent absorption of the substance by the oral and the inhalation route no systemic hazard can be expected for repeated inhalation route of exposure. Moreover, no significant inhalation exposure is expected.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One reliable 28d-study available

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No data on inhalation toxicity after repeated exposure is available as the corresponding test was waved based on exposure and considerations. No significant inhalation exposure is expected as the substance is an organic salt and has a very low vapour pressure and a high melting point. The potential for the generation of inhalable forms is considered to be low. The use of this substance will not result in aerosols, particles or droplets of an inhalable size, so the exposure to humans via inhalation will be unlikely to occur. Even if long-term inhalation exposure with the substance would occur, it would be considered to be not hazardous, assuming similar 100 % absorption for oral and inhalation route.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No data on inhalation toxicity after repeated exposure is available as the corresponding test was waved based on exposure and considerations. No significant inhalation exposure is expected as the substance is an organic salt and has a very low vapour pressure and a high melting point. The potential for the generation of inhalable forms is considered to be low. The use of this substance will not result in aerosols, particles or droplets of an inhalable size, so the exposure to humans via inhalation will be unlikely to occur. Even if long-term inhalation exposure with the substance would occur, it would be considered to be not hazardous, assuming similar 100 % absorption for oral and inhalation route.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
A skin penetration test (see IUCLID Chapter 7.1.2) shows very low penetration of the test material (5% in 24 hours). In view of the absence of repeated oral dose toxicity (see IUCLID Chapter 7.5.1: NOAEL rat oral (28d) >= 1000 mg/kg bw/day) and the low skin penetration of the substance systemic toxicity is unlikely to occur from repeated dermal exposure. Further, the substance is neither irritating nor sensitising to skin.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
A skin penetration test (see IUCLID Chapter 7.1.2) shows very low penetration of the test material (5% in 24 hours). In view of the absence of repeated oral dose toxicity (see IUCLID Chapter 7.5.1: NOAEL rat oral (28d) >= 1000 mg/kg bw/day) and the low skin penetration of the substance systemic toxicity is unlikely to occur from repeated dermal exposure. Further, the substance is neither irritating nor sensitising to skin.

Justification for classification or non-classification

Based on the available data the substance is not hazaordous when exposed repeatedly via oral, dermal or inhalation route, neither for any local effect nor regaring systemic adverse effects. Therefore, a classification according to EC 1272/2008 is deemed to be not necessary.