Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Triisodecyl phosphite used for read across was tested in an OECD 422 modified test. It was carried out with CD rats by daily gavage up to 1000 kg/kg bw/d. No treatment related effects were found for parental toxicity effects, reproductive toxicity or offspring toxicity. The results of the studies were as follows:

NOAEL reproductive toxicity ≥ 1000 kg/kg bw/d

NOAEL offspring toxicity ≥ 1000 kg/kg bw/d

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
quoted Klimlisch 1 in the frame of US HPV dossier in 2001, then quoted 2 for read across approach
Justification for type of information:
see Read-across justification
Reason / purpose:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
2 weeks of prebreed exposure (males and females), 2 weeks of mating (males and females) and 3 weeks of gestation and lactation each (F0 females) (daily
No systemic toxicity was shown at any dose at any time.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology; FOB
No reproductive toxicity was shown in the offspring generation.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight and external and visceral examination
Reproductive effects observed:
not specified
Conclusions:
Under the test conditions, the NOAELs for F0 reproductive toxicity were observed at or above 1000 mg/kg/day for males and females. The NOAELs for F1 offspring toxicity during lactation were also at or above 1000 mg/kg/day for males and females.
Executive summary:

Triisodecyl phosphite administered by gavage once daily at 0, 50, 250 and 1000 mg/kg/day to parental F0 CD (SD) rats, 10/sex/group through prebreed, mating, gestation and F1 lactation resulted in essentially no treatment or dose related adult F0 parental toxicity at any dose at any time. Reproductive toxicity was not present in F0 males or females. There was also no F1offspring toxicity observed postnatally through the weanling necropsy. Therefore, the F0 male and female systemic no observable adverse effect level (NOAEL) was at or above 1000mg/kg/day for males and females. The NOAELs for F0 reproductive toxicity were observed at or above 1000 mg/kg/day for males and females. The NOAELs for F1 offspring toxicity during lactation were also at or above 1000 mg/kg/day for males and females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The possible adverse effects of the Phosphorous acid, tri-C12-14-alkyl esters on pregnant rat females and rat embryo-foetal development via oral administration (gavage), during gestation days 5 to 19, was assessed according to the OECD guideline 414. The study was GLP compliant.

The 4 groups of 25 female Wistar rats each were exposed to the following doses : 0 (control), 100, 300 and 1000 mg/kg bw/day (Low Dose, Medium Dose and High Dose).

No adverse systemic effects of the test item were found up to 1000 mg/kg bw/day and no treatment related effects were found for both maternal toxicity and foetal toxicity. The NOAEL for both maternal and foetal toxicity in this study is ≥ 1000 mg/kg bw/day.

Moreover the study was carried out according to the recently updated OECD guideline (June 2018) including additional observations to detect potential endocrine disruptive properties but no finding in the study supported a possible endocrine disruption modality of the test item.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
july 2018 till december 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
yes
Remarks:
These deviations did not influence the quality or integrity of the present study.
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age of the females at the start of pairing: at least 12-13 weeks old
- Weight before initiation of pairing: males: 309 - 377 g (mean: 352.96 g, ± 20 % = 282.37 – 423.55 g) and females: 194 - 246 g (mean: 220.34 g, ± 20 % = 176.27 – 264.41 g)
- Fasting period before study:
- Housing: The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (up to 5 animals / cage) in type IV cages.
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimation period (at least 5 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Based on the results of stability testing , the test item formulations were prepared at least once every 5 days (within stability time frame as given by a stability and homogeneity Study). The prepared formulation was stored protected from light and at 2-8 °C. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.

ADMINISTRATION OF DOSES:
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 4 mL/kg body weight with different concentrations (LD= 25 mg/mL / HD= 75 mg/mL / HD = 250 mg/mL)
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics and testing guideline.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at biochemA GmbH as part of a separate GLP study.
The pre-study for stability analysis included samples from the low, mid and high dose groups and the investigation was carried out for 10 days at 20 ± 5 °C (RT – room temperature), 10 days at 5 ± 3 °C (2-8 °C) and 10 days at -20 ± 5 °C.
Prestart homogeneity investigation included samples collected from various levels (top, middle and bottom) of the high, medium and low dose groups. The test item was shown to be homogenous according to the stability study (after 30 min without stirring). Therefore, samples were not collected during the study for the investigation of homogeneity; samples were only taken for substance concentration checking in the first and last weeks of the study for all doses (8 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B,each of 3 mL).
The A-samples were investigated two or three days after sampling. Analytical samples were stored at -20±5 °C
before and after measurement. The B-samples were retained and discarded after completion of the final study report.
Details on mating procedure:
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the mating a detailed clinical observation outside the home cage was made. Only healthy animals were used for this study.
Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. 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 gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body
weights were comparable with each other. Each animal was assigned a unique identification number. After getting 100 sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week between gestation day 5 until gestation day 19.
Frequency of treatment:
The test item was administered daily.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
LD = low dose,
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
MD = medium dose,
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
HD = high dose
No. of animals per sex per dose:
4 groups of 25 female Wistar rats each
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were chosen according to the results of the dose range finding study (BSL Munich Study No. 178742.non-GLP) . The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
All animals were weighed once before initiation of pairing to ensure that the body weights were within ± 20 % variation.
The sperm positive females were weighed on 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 : Yes
Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION : No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20: On gestation day 20, sperm positive females were subjected to a caesarean section after sacrificing the animals using anesthesia (ketamine/xylazine).
- At the time of termination or death during the study, the dam (presumedly pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4 % neutral-buffered formaldehyde.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear nongravid 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.

EVALUATION OF THYROID HORMONES: Yes
Thyroid hormones 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 stored under appropriate conditions. Blood samples were assessed for serum levels for thyroid hormones (T3, T4, TSH).

HISTOPATHOLOGY:
A full histopathology was carried out on the preserved thyroid/parathyroid glands from all dams of all dose groups which were sacrificed at the end of the treatment period.
Ovaries and uterine content:
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. 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 foetuses in each uterine horn was also recorded.
Fetal examinations:
All foetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies and the anogenital distance (AGD) of each foetus was measured.
Foetal body weight measured on gestation day 20 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of foetus weight).
One half of each litter was examined for soft tissue anomalies by a microdissection technique.
The remaining foetuses were processed by Alizarin staining and the first 20 litters per group were examined for skeletal alterations.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination of the first 20 litters per group were performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

- External Examination:
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout were examined for the shape and size.
The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

- Skeletal Examination:
Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95 % ethanol. These foetuses were processed using the Alizarin red staining technique.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centers.
The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

- Visceral Examination:
The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off center and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for their presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.
The lung was observed for size, colour and number of
lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula. The position, size, colour and shape of the heart was recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle into the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.

- Craniofacial Examination:
A single foetus was decapitated and the head of the fetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights and thyroid hormones were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics was performed with 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):
Salivation and moving the bedding were two clinical signs that were seen transiently in timely relation to dose administration and were considered as slight clinical signs elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to the oral administration, but not systemic toxicity.
Piloerection was observed in 2/25 females of the control group, 1/25 females of the LD group, 2/25 females of the MD group, and 1/25 females of the HD group. Piloerection was seen without dose-dependency and was considered as a sign of discomfort but not systemic toxicity.
Slight clinical signs like a bloody toe in 1/25 females of the control group, crusts in 1/25 females of the LD and 1/25 females of the HD group, hairless areas in 2/25 females of the LD group, 1/25 females of the MD group, and 2/25 females of the
HD group, and regurgitation of the vehicle in 1/25 females of the control group were noted without dose-dependency and were not considered toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in any of the groups in the course of this study. All animals of the test item-treated groups and the control group survived the scheduled study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no biologically relevant differences in body weight development when comparing the test item-treated groups to the control group. Intermittently, statistically significantly higher body weight gain in the LD (24,48g) and HD (27.19g) group compared to the control group (20.04g) from GD 14-17 was observed. However, this was not considered toxicologically relevant due to the absence of dose-dependency and the inconsistency of this observation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by the treatment with the test item. Slight but statistically significantly higher (88.81 g in the HD group compared to 80.33 g in the control group) food consumption in the HD group (GD 0-5) compared to the controls was not considered toxicologically relevant as animals were not treated with the test item from GD 0-5. Other dose groups showed no differences compared to the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Description (incidence and severity):
See clinical signs
Immunological findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Few macroscopic changes were recorded in female animals and they were not considered to be of test item treatment relevance.
Indeed the macroscopic changes observed at necropsy were a yellow mass in the tissue surrounding fat of the ovary in HD female no. 94, a hard and dark mass in the tissue surrounding fat of the uterus in female no. 50 of the LD group, and an enlarged placenta of female no. 17 of the control group. These findings had low incidences, followed no dose-dependency and were considered incidental.
Relative and absolute mean thyroid/parathyroid gland weight showed no statistically significant differences when comparing test item-treated groups to the control group. Without correlating findings in the histopathological evaluation of the thyroid/parathyroids, the slight differences observed between the groups were not considered toxicologically relevant.
Neuropathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant gross pathological findings at necropsy and no morphological lesions in the thyroid/parathyroid glands.
No test item-related effect of statistical significance or toxicological relevance was observed on maternal thyroid hormone (T3, T4, TSH) levels in the test item-treated groups when compared to the controls. Levels of measured hormones were comparable between all groups.
Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There were no dose-dependent, toxicologically relevant differences for pre- and post-implantation loss in the test item-treated groups when compared to the control group. However, slightly higher pre-implantation loss in the
control and in the LD groups was observed when compared to the MD and HD group (8.5 % in the control group compared to 4.7 % in the LD, 1.4 % in the MD and 1.4 % in the HD groups). Slightly higher values of pre-implantation loss of the control and LD group were mainly caused by high values of single females (female nos. 5 and 23 in the control group and female no. 50 in the LD group). Mean values of the control and LD group were within the normal range of variation and were not considered toxicologically relevant as higher dose groups were not affected.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Prenatal parameters like early and late resorptions remained unaffected in the dose groups when compared to the control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were observed in any of the test item-treated groups or the control group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 25/25 pregnancies in the LD group, 20/25 pregnancies in the MD group and 21/25 pregnancies in the HD group and compared to 24/25 pregnancies in the control group. This pregnancy rate was considered to be within
the normal range of this rat strain and age.
Other effects:
no effects observed
Description (incidence and severity):
There were no biologically relevant or statistically significant effects on prenatal data.
Prenatal parameters like number of corpora lutea, implantation sites, live foetuses, early and late resorptions, and sex ratio remained unaffected in the dose groups when compared to the control group.
Details on maternal toxic effects:
No biologically relevant or statistically significant differences were observed in mean maternal terminal body weight, gravid uterus weight and resulting adjusted maternal weight when comparing test item-treated groups and the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Abnormalities:
no effects observed
Localisation:
ovary
oviduct
uterus
cervix
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean foetal weight showed no toxicologically relevant effects in the test item-treated groups when compared to the control group.
Related to the individual foetuses, mean foetus weight was marginally but statistically significantly higher in the HD group when compared to the control group (7 %). Without corresponding findings in any of the other parameters like number of males, females and total number of foetuses, these differences were considered as biological variation and not related to the treatment with the test item.
Mean foetus weight was observed to be slightly but statistically significantly higher in male foetuses and female foetuses of the HD group compared to foetuses of the control group (8 % and 7 % above controls, respectively). However, mean foetus weight was well within the range of historical control data and slightly higher mean foetus weight in the HD group was not considered of toxicological relevance. Along with higher mean weight, absolute and relative anogenital distance was statistically significantly higher in female foetuses of the HD group compared to female foetuses of the control group (9% and 7 % above controls, respectively). This can be attributed to the higher weight of foetuses of the HD group and is not assumed to be toxicologically relevant.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Prenatal parameters like live foetuses remained unaffected in the dose groups when compared to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Prenatal parameters like sex ratio remained unaffected in the dose groups when compared to the control group.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Mean litter weight showed no toxicologically relevant effects in the test item-treated groups when compared to the control group.
However, statistical analysis of weight data on a per litter basis showed slightly but statistically significant higher total litter weight in the HD group compared to the control group (20 %). As total litter weight is based on the litter size and litters of the HD group were slightly bigger (mean total no. of fetuses: 10.81) compared to the control group (mean total no. of foetuses: 9.67), higher litter weight was not considered of toxicological relevance.
External malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities considered to be of toxicological relevance in any of the test item-treated groups. Statistical analysis of litter incidences showed no significant differences compared to the control group.
By external examination, an umbilical hernia was noted in one foetus of the control group and was considered as spontaneous in nature.
Haematoma were observed in one single foetus of the control group (on neck) and one single foetus of the HD group (on right hindlimb). Due to the low number of incidences, the lack of dose dependency and the occurrence of a haematoma also in the control group, these findings were not considered toxicologically relevant.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination by revealed few findings (subcutaneous head oedema, dilated third ventricle, dilated lateral ventricle, retinal fold and small/misshapen pituitary gland) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls without any dose-dependent trend. Statistical analysis of the data revealed no significant effect in any of the mentioned investigated areas. These findings were considered to be spontaneous in nature and not related to the treatment with the test item.
Slight but statistically significant higher occurrence of percent litter incidence for subcutaneous forebrain oedema was observed in the HD group when compared to the control group (20% litter incidence in the HD group compared to 0% litter incidence in
the control group). In the light of the fact that just 4/102 foetuses from 4 litters were observed with this finding and that no corresponding external findings like enlarged head was observed in these fetuses this finding was not considered to be adverse.

Skeletal Examination:
Skeletal examination o revealed a range of findings which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group. None of these findings achieved statistically significant differences when comparing foetuses of test item-treated groups to foetuses of the control group.
There were a few skeletal findings including incomplete ossification of a few bones and a few other skeletal findings observed in the HD group without achieving statistical significance:
- Slightly higher litter incidences, but without achieving statistical significance, were observed in the HD group compared to the controls for increased ossification in the orbital socket region (95 % compared to 80 % in the control group), increased
ossification of auditory ossicles (45 % compared to 15 % in the control group), irregular ossification of centra of thoracic vertebrae (50 % compared to 40 % in the control group), irregular ossification of cervical vertebral arches (65 % compared to 45 % in the control group), fusion of centra and arches of caudal vertebrae (65 % compared to 40% in the control group), increased ossification of hindlimb calcani 5 % compared to 0 % in the control group), and increased ossification of hindlimb metatarsals (75 % compared to 45 % in the control group). The observed incomplete ossification of a few bones and a few other skeletal findings in the HD group were either marginally higher or within historical control data range (without achieving statistical significance). Generally delayed ossification is not regarded to persist postnatally and is not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.
- Foetuses of the HD group were observed with aslightly higher incidence of fused arches and centra of sacral vertrebrae when compared to control foetuses (80 % in the HD group compared to 45 % in the control group). However, this difference achieved no statistical significance and together with the lack of dose-dependency was not considered test item-related. Foetuses of all test item-treated groups showed a slightly higher incidence of fused centra and arches of sacral vertebrae when compared to control foetuses (65 % in the LD group, 55 % in the MD, and 80 % in the HD group compared to 45 % in the control group).
- The finding of rudimentary ribs showed a slight correlation between incidences and test item-dose without achieving statistical significance in any of the groups compared to the controls. Litter incidence was 60 % in the control group, 70 % in the LD group, 75 % in the MD group, and 80 % in the HD group. Rudimentary/short ribs are considered as transient abnormalities. In contrast, full/long ribs are considered permanent and may cause health effects in humans. However, postnatal consequences in rats are unknown but are assumed to be minimal [12] [13]. The incidence of full ribs (bilateral) was marginally higher in the HD groups compared to the controls (15 % compared to 20 % in the control group).
- Wavy ribs were observed with a litter incidence of 45 % in the control group, 60 % in the LD group, 65 % in the MD group, and 50 % in the HD group and thus followed no dose-dependent trend. The differences between the test item groups and the control group did not achieve statistical significance. Wavy ribs are common findings in rodent studies and are considered to be postnatally reversible.Thus, wavy ribs are classified as variations and were not considered as an adverse effect of the treatment with the test item.

These findings showed no dose-dependency and were within the historical control data range and thus were not considered to be adverse.
Visceral malformations:
no effects observed
Description (incidence and severity):
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all test item-treated groups and the control group. Affected organs/tissues were the umbilical artery (transposed), aortic arch (dilated), ductus arteriosus (dilated), heart (fluid-filled pericardium), liver (discolored, spotted, supernumerary lobe, cleft), testis (discolored), lung (ragged border: completely), ovaries (cyst), ureter (convoluted, dilated), uterus (dilated), kidneys (cyst, discolored, dilated renal pelvis), urinary bladder (cyst, discolored), adrenal glands (discolored, spotted), thymus (discoloured, large, long) and spleen (discoloured, large).
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. Litter incidences were not statistically significant.
Observed findings were either minor variation and/or considered to be spontaneous in nature. Due to a lack of dose dependency and consistency they were not considered of toxicological relevance.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Dose Formulation Analysis:

Mean accuracy of analytical samples ranged from 113.3 to 117.1 % with a COV<4.3% on the first sampling and from 114.4 to 122.0 % with a COV<4.4 % on the second sampling.

Measured concentrations were slightly above the nominal values for all doses.

Higher concentrations in stability samples were also observed during validation. The obtained result can be caused by the larger scale of the sample preparation.

This does not affect the integrity of the study data.

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

The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of female rats to Phosphorous acid, tri-C12-14-alkyl esters via oral administration (gavage) during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0).

The study was performed according to the OECD 414 guideline and was GLP compliant.

The 4 groups of 25 female Wistar rats each were exposed to the following doses 0 (control), 100, 300 and 1000 mg/kg body weight/day (Low Dose, Medium Dose and High Dose). Animals of the control group were handled identically as the dose groups, but received corn oil, the vehicle used in this study. The test item was administered daily from gestation day 5 to gestation day 19 to female animals. Dose volumes were adjusted individually based on weekly body weight measurements.

During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. All female animals were sacrificed on the respective gestation day 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), live and dead foetuses. Foetuses were identified by color strings, sexed and weighed. All foetuses were observed for external abnormalities, half of the foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities.

The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20.

Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.

The uteri of the non-pregnant females were checked for the early embryonic deaths.

The weight of thyroid/parathyroid glands was measured and blood samples were assessed for serum levels for thyroid hormones (T3, T4, TSH). A full histopathology assessment was carried out on the preserved thyroid/parathyroid glands.

On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with Phosphorous acid, tri-C12-14-alkyl esters, 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.

Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in any of the test item-treated groups. However, there were a few skeletal findings including incomplete ossification of a few bones and a few other skeletal findings observed without achieving statistical significance in the HD group. These findings showed no dose-dependency and were within the historical control data range and thus were not considered to be adverse effects caused by the test item.

No finding in the study supported a possible endocrine disruption modality of the test item.

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

None of the repeated dose toxicity studies available showed any reproductive toxicity alert. No classification is then required at the moment.