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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 414
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methylphosphonic acid, compound with amidinourea (1:1)
EC Number:
282-758-4
EC Name:
Methylphosphonic acid, compound with amidinourea (1:1)
Cas Number:
84402-58-4
Molecular formula:
C2H6N4O.CH5O3P
IUPAC Name:
methylphosphonic acid, compound with amidinourea (1:1)
Details on test material:
technical product

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Applicant's summary and conclusion

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.