Phosphinylidynetrimethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 18 July 2020 - 24 September 2020 (last necropsy)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 18 July 2020 - 24 September 2020 (last necropsy)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted July 29, 2016

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"; deviations are not affecting the reliability of the study

Principles of method if other than guideline:

Due to technical reasons, the frozen plasma samples for the optional T4, T3 or TSH analyses were stored at -70 °C instead of -80 °C (nominal values), as indicated in the Study Plan.
Due to technical reasons, blood sampling from animal 3504 could not be performed; therefore, an additional animal (3508) from the same dose group was used for clinical chemistry and urine evaluation.
These deviations are considered to have no impact on the outcome of the study or on interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Budapest, Hungary
- Age at study initiation: approximately 10 weeks
- Body weight range at study initiation: 316 to 421 g (males); 211 to 253 g (females)
- Fasting period before study: no
- Housing in T3H polycarbonate cages: The animals were group-housed, up to 3 animals of the same sex/cage, with the exception of the mating and gestation/delivery/lactation period, when they were paired or individually housed (with pups), respectively.
- Diet: standard laboratory rat diet, ad libitum [SM Rat/Mouse, Breeding & Maintenance, 15 mm, autoclavable; manufacturer: ssniff Spezialdiäten GmbH, Germany)
- Water: regularly monitored tap water from the municipal supply, as for human consumption, from drinking bottles, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 – 23 °C
- Humidity: 42 - 69 %
- Air changes: 15 to 20 per hour
- Photoperiod: 12 hours light (6:00 - 18:00) and 12 hours dark

IN-LIFE DATES: 2020-07-18 to 2020-09-24 (necropsy of last animals)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test item was formulated in the vehicle (distilled water), as an aqueous solution at the appropriate concentrations. Formulations were prepared up to 8 days before use and were kept at room temperature until use. The formulation samples in the 1-220 mg/mL concentration range (using distilled water as vehicle) were proven as being stable for at least 21 days when stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: As determined in pre-tests, water was was selected as vehicle for this study.
- Concentration in vehicle: 1-220 mg/mL
- Amount of vehicle: 5 mL dose preparation/kg body weight
- Purity: 100% (distilled water, pharmaceutical grade Ph.Hg.VIII)

Details on mating procedure:

- M/F ratio per cage: 1 / 1
- Length of cohabitation: Each female was placed with the same male until copulation occurred.
- Proof of pregnancy: A vaginal smear was prepared daily during the mating period and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy).
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test item formulations for concentration and homogeneity was performed at the test site. Top, middle and bottom duplicate samples were taken from the test item formulations three times during the study (during the first and last weeks and approximately midway during the dosing period), one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected samples were stored at room temperature until shipment. Samples (one set) were shipped as soon as possible after collection for concentration and/or homogeneity measurement to the Principal Investigator (PI).
The formulation analyses were conducted under the control of the PI. Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration. Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) should be less than 10%.
The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirm that the formulations were homogeneous and stable from the time of preparation until completion of dosing.

Duration of treatment / exposure:

- Parental males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
- Parental females were dosed for 14 days pre-mating, for up to 6 days during mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition is complete) was defined as Day 0 post-partum. Females showing no evidence of pregnancy were sacrificed as practical (27-30 days after evidence of copulation).

Frequency of treatment:

Once daily (7 days / week)

Details on study schedule:

- Animal age at mating in the study: 14 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on the results of a oral gavage dose-range finding (DRF) study with THPO in the rat performed at the same test facility and using the same species and strain - where no clear test item related effects were seen up to 1000 mg/kg bw/day - doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Positive control:

No positive control

Parental animals: Observations and examinations:

- Clinical observations: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
General clinical observations were made once a day.
Detailed clinical observations were made on all animals outside the home cage in a standard arena at the start of the pre-exposure, prior to the first dosing (to allow for within-subject comparisons) and at least weekly thereafter, in the morning hours. Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc.), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

- Functional observation battery (FOB): Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 24-26; females on PPD 9-11). In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement.
Five males and five females per group were subjected to the functional observation battery, including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.
To measure the landing foot splay, the fore and hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots of the hind limbs were measured (the distance of the two forelimbs was not calculated as it was not deemed necessary by the Study Director).
Fore/hind grip strengths were measured using a grip strength meter (Nova ABF-50 Advanced with custom adapters). The rats were held appropriately such that the fore limbs allowed to grip the support bar and gently pulled back until they release the bar; the device measured the maximum grip strength. This was performed 3 times for each animal. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
Locomotor activity was recorded by placing the animals individually into an open-field for at least 1-hour observation time. Recording was made for a duration of at least 45 minutes, under dim-light and undisturbed conditions. The recorded video was processed and the calculated data was evaluated for distance travelled in 5 minute segments.

- Body weight measurements: Body weights of all parental animals were recorded with a precision of 1 g weekly during the pre-exposure period, on the first day of dosing (Day 0, prior to start of dosing), then afterwards at least weekly and prior to the scheduled necropsy (fasted).
Parental females were weighed on GD 0, 3, 7, 10, 14, 17, 20, and on PPD 0 (within 24 hours after parturition), 4, 7, 10, 13, and at termination (PPD 14, fasted). The body weight of the female animals measured on GD 3, 10 and 17 as well as PPD 7 and 10 were only additional measurements as aid for the calculation of accurate dosing volumes, and thus are not reported.

- Food consumption measurement: The determination of food consumption was performed weekly (on body weight measurement days). The remaining, non-consumed food was weighed with a precision of 1 g. Daily food consumption was calculated.

- Oestrus cycle monitoring: Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the dosing started. Vaginal smears were also checked daily from the beginning of the dosing period until evidence of mating (during the pre-mating and mating periods). Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

- Observation of the delivery process, offspring and nursing instinct: Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. Any evidence of abnormal deliveries were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy.
Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring were recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4 and PND 13, with accuracy of 0.01 g. All the litters were checked and recorded daily for the number of viable and dead pups, any abnormal behaviour or appearance of the pups was also recorded.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). The anogenital distance were also normalized to the pup size (the cube root of body weight). Number of nipples/areolae in male pups were recorded on PND 13.
One male and one female pup per litter (if possible) were selected for culling for blood sampling on PND 4. After that, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter. No pups were eliminated when litter size drooped below the culling target (10 pups/litter).
All remaining F1 offspring were terminated on PND 13. (In order to allow for overnight fasting of dams prior necropsy on PND 14, offspring were euthanized on PPD/PND 13, and the dams on PPD/PND 14.)

The following parameters were reported:
- Parental males:
Clinical observations and functional observation battery (FOB)
Body weight and body weight gain
Food consumption
Number of pairings
Number of fertile pairings
Number of infertile males
Male mating index
Male fertility index
Clinical pathology (including T4 hormone analysis)
Gross necropsy findings
Organ weights
Histopathology findings

- Parental females:
Clinical observations and functional observation battery (FOB)
Body weight and body weight gain
Food consumption
Oestrus cycle data (including number of females with normal and abnormal oestrous cycles)
Number of pairings
Number of pregnant females
Number of sperm positive, but non-pregnant females
Number of non-mated females
Female mating index
Female fertility index
Gestation index
Duration of pregnancy (in days)
Number of implantation sites / dams
Number of dams with live pups on Day 0, 4 and 13
Intrauterine mortality
Total offspring mortality (intra- and extrauterine mortality)
Clinical pathology
Gross necropsy findings
Organ weights
Histopathology findings

- Offspring:
Mean pup body weight (per pup within the group and per litter) on PND 0, 4 and 13
Mean pup body weight gain (per litter) between PND 0-4, 4-13 and 0-13
Number of live births per litter, and number of viable pups per litter on PND 0, 4 and 13
Survival index of pups on PND 0, 4 and 13
Sex ratio (for females) on PND 0, 4 and 13
T4 hormone analysis
Anogenital distance
Nipple retention
Thyroid glands weights

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the dosing started. A vaginal smear was prepared daily during the mating period and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The efficiency of suckling was observed by the presence of milk in the pups' stomach.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring were recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4 and PND 13, with accuracy of 0.01 g. All the litters were checked and recorded daily for the number of viable and dead pups, any abnormal behaviour or appearance of the pups was also recorded.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). The anogenital distance were also normalized to the pup size (the cube root of body weight). Number of nipples/areolae in all pups were recorded on PND 13.
One male and one female pup per litter (if possible) were selected for culling for blood sampling on PND 4. After that, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter. No pups were eliminated when litter size drooped below the culling target (10 pups/litter).
All remaining F1 offspring were terminated on PND 13. (In order to allow for overnight fasting of dams prior necropsy on PND 14, offspring were euthanized on PPD/PND 13, and the dams on PPD/PND 14.)

GROSS EXAMINATION OF DEAD PUPS: yes
Dead pups and pups euthanized on PND 4 or PND 13 were carefully examined externally for gross abnormalities. Presence of nipples/areolae in the PND 13 pups was also recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 14 days mating/post-mating period
- Maternal animals: All surviving animals after PPD/PND 14

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in section "overall remarks" were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Dead pups and pups euthanized on PND 4 or PND 13 were carefully examined externally for gross abnormalities. Presence of nipples/areolae in the PND 13 pups was also recorded.

Statistics:

Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables. Frequency and percentage were calculated for categorical variables in Microsoft Excel.
Statistical analysis was performed for the continuous variables using an automated decision tree within the R software. The following decision tree was applied:
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Reproductive indices:

- Mating index (measure of animals’ ability to mate)
- Fertility Index (measure of male's ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant)
- Gestation Index (measure of pregnancy that provides at least one live pup)

Offspring viability indices:

- Survival index of pups on PND 0, 4 and 13
- Sex ratio (for females) on PND 0, 4 and 13
- Mean pup body weight gain (per litter) between PND 0-4, 4-13 and 0-13

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was seen during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the body weight and body weight gain parameters of the test item treated animals in any of the sexes when compared to control data. The occasional statistically significances are considered incidental, not test item related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed on food consumption of the test item treated animals in any of the sexes when compared to control data. The statistically significant values in the male High dose group at the Day 14-21, 21-27 and 0-27 periods, and in the female Low dose group at the GD 14-20 period are considered incidental, toxicologically not relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed in the haematology parameters.
Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed in the clinical chemistry parameters.
Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the urinalysis parameters.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of grip strength, landing foot splay or locomotor activity.
All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. Occasional statistical significance for an individual segment without a clear trend or dose response was not considered as test item related effect.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related microscopic changes were observed in the investigated reproductive and other organs of the animals.
The acute hemorrhage in the mandibular lymph node and thymus is considered as consequence of hypoxia, dyspnoe and circulatory disturbance developed during exsanguinations, without pathological significance.
Histological examination revealed in the stomach mucous membrane circumscribed congestion. No erosion, ulceration or inflammation was observed. It is considered to be caused by a mechanical effect, most probably related to the oral gavage of the animals.
The cyst in the kidney is considered as an individual disease.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The cyst in the kidney is considered as an individual disease.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods.
Prolonged diestrus (presumably pseudopregrancy) was recorded for one High dose female, but then the cycles of the animal became normal and the animal successfully mated and became pregnant. This is considered as being a normal finding, not being a test item related effect.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test item related microscopic changes were observed in the investigated reproductive and other organs of the animals.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item dosed groups with regard to reproductive ability, mating or gestation indices, that could be ascribed to the test item administration. The mating index was 100% in all groups (males and females). In the Low dose one female, and in the Mid dose 4 females did not get pregnant out of the 12 animals, which is considered normal. Also, no dose-dependency was observed. All females in the Control and the High dose groups became pregnant and delivered healthy litters.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation). The mean

- Mortality: No mortality was seen during the study.

- Clinical observation: No clinical signs were observed in the study.

- Body weight and body weight gain: No test item related changes were observed in the body weight and body weight gain parameters of the test item treated animals in any of the sexes when compared to control data. The occasional statistically significances are considered incidental, not test item-related.

- Food consumption: No test item related changes were observed on food consumption of the test item treated animals in any of the sexes when compared to control data. The statistically significant values in the male High dose group at the Day 14-21, 21-27 and 0-27 periods, and in the female Low dose group at the
GD 14-20 period are considered incidental, toxicologically not relevant.

- Neurological assessment: There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, landing foot splay or locomotor activity. All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. Occasional statistical significance for an individual segment without a clear trend or dose response was not considered as test item related effect.

- Haematology: No test item-related changes were observed in the haematology parameters. Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

- Clinical chemistry: No test item-related changes were observed in the clinical chemistry parameters. Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

- Thyroid hormone analysis (adult): No effect of test item was observed in the thyroid hormone analysis. Compared to the control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males. Because of this, no further investigation was performed on the thyroid hormones (i.e. assessment of samples collected from the dams, assessment of other hormones, etc.).

- Urinalysis: No test item-related changes were observed in the urinalysis parameters.

- Organ weights: No treatment-related effects were observed in the experimental animals. There were no biologically relevant differences among groups in the weights of organs measured when compared to Controls in any sexes.The absolute brain weight in the male High dose group and the relative (to the body weight) kidney and uterus weights in the female High dose group were statistically significantly different from the controls. These observed means were well within the historical control ranges in all cases, there were no clear dose responses and/or values were without any similar trend in the other sex of animals. Furthermore, there was no histopathological correlate for the weight changes. Thus, they were considered as having no toxicological relevance.
 
- Pathology evaluation (macroscopic findings): No test item-related macroscopic changes were observed in the terminally euthanized animals. The following findings were recorded at necropsy: Dark red diffuse discoloration of the mandibular lymph nodes in 1/12 male Control, 2/12 male Low dose, red/dark red diffuse discoloration of the thymus in 1/12 male High dose and 1/12 female High dose animals; dark red single focal or multifocal discoloration of the glandular mucosa of the stomach was observed in 3/12 female Control, 2/12 female Low dose, 3/12 female Mid dose and 4/12 female High dose animals; in one High dose female a cyst was observed on the right kidney, and also the left kidney was enlarged and the liver had multifocal dark red discoloration on all lobes.

- Histopathology evaluation (microscopic findings): No test item-related microscopic changes were observed in the investigated reproductive and other organs of the animals. The acute hemorrhage in the mandibular lymph node and thymus is considered as consequence of hypoxia, dyspnoe and circulatory disturbance developed during exsanguinations, without pathological significance. Histological examination revealed in the stomach mucous membrane circumscribed congestion. No erosion, ulceration or inflammation was observed. It is considered to be caused by a mechanical effect, most probably related to the oral gavage of the animals. The cyst in the kidney is considered as an individual disease.

- Oestrus cycle evaluation in females / pre-exposure period: Each female selected for the study showed acceptable cycles before starting the dosing period. One animal had prolonged diestrus (presumably, it was the end of a pseudopregnancy) in the beginning of the pre-exposure period, but then the oestrus cyclicity became normal and thus the animal was not excluded from the study.
 
- Oestrus cycle evaluation in females / exposure period (pre-mating and mating periods): No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods. Prolonged diestrus (presumably pseudopregrancy) was recorded for one High dose female, but then the cycles of the animal became normal and the animal successfully mated and became pregnant. This is considered as being a normal finding, not being a test item related effect.

- Reproductive ability assessment and indices: There were no differences between the control and test item dosed groups with regard to reproductive ability, mating or gestation indices, that could be ascribed to the test item administration. Both the mating and fertility index was 100% in all groups (males and females). In the Low dose one female, and in the Mid dose 3 females did not get pregnant out of the 12 animals, which is considered normal. Also, no dose-dependency was observed. All females in the Control and the High dose groups became pregnant and delivered healthy litters. Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 6 days of pairing (cohabitation). The mean duration of mating was 2.83, 2.64, 2.30 and 2.42 days in the Control, Low, Mid and High dose groups, respectively.

- Evaluation of the pre-natal and gestation periods: There was no effect of treatment noted during the gestation period, parturition and postpartum period in any of dose groups. The mean duration of pregnancy was comparable in the Control and test item treated groups. As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted. The number of implantation sites was comparable to the control mean in all dose groups. There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values in any dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values in any of the dose groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 were comparable to control values in each dose group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls. The measured values were within the range commonly recorded for this strain and age.
When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND0, 4 and 13 showed no toxicologically significant differences compared to controls in the F1 generation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the absolute or normalized anogenital distance measured on PND 0 were noted for test item treated male and female pups, when litter mean values were compared to control.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no nipples/areolae presence seen in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

- Mortality and clinical observations: There was no test item effect on mortality or survival of the pups (F1 generation). The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 were comparable to control values in each dose group. There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values in any of the dose groups. Evidence of suckling was recorded for all live born pups in the study. The sex ratio of female pups was comparable to the Control in each dose group on PND0, PND4 and PND13.

- Body weight and body weight gain: There were no test item related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls. The measured values were within the range commonly recorded for this strain and age. When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND0, 4 and 13 showed no toxicologically significant differences compared to controls in the F1 generation.

- Anogenital distance, nipple retention: No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the absolute or normalized anogenital distance measured on PND 0 were noted for test item treated male and female pups, when litter mean values were compared to control. There was no nipples/areolae presence seen in any of the male pups on PND13.

- Thyroid gland weights and T4 hormone levels: No test item related differences were observed in the absolute or relative thyroid weights of the F1 generation, compared to control. The statistical significance in the Mid dose group is considered incidental, not related to the test item. The mean T4 level of the PND13 pups was comparable in the Control and test item treated groups. The slightly higher value in the Mid dose group, reaching statistical significance, is considered to be biologically relevant.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The study was performed acc. to OECD TG 422 under GLP, so the results can be considered reliable. The no observed adverse effect level (NOAEL) for systemic, and reproductive and developmental toxicity is 1000 mg/kg/day under the conditions of this study. The test item is considered not to exhibit any toxicologically relevant effects up to the limit dose of 1000 mg/kg.

Executive summary:

The reproductive toxicity of Tris(hydroxymethyl)phosphine oxide (THPO) was investigated in a Reproduction/Development Toxicity Screening study following repeated daily administration by oral gavage to Wistar rats, conducted in accordance with the standardised guideline OECD 422, under GLP conditions.

The objective of the study was to screen for possible effects of the substance on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD13.

The test item was administered orally (by gavage) at 0, 100, 300 or 1000 mg/kg bw/day, corresponding to concentrations of 0, 20, 60 and 200 mg/mL at a 5 mL/kg bw dose volume to four dose groups of Wistar rats (n=12/sex/group) once a day.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessments, such as functional observation battery (FOB) including measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offsprings until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. A detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed.

In summary, daily administration of Tris(hydroxymethyl)phosphine oxide (THPO) by oral gavage to Hannover Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in mortality or any clinical signs.

The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.

At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.

No test item-related findings were noted in the clinical pathology parameters.

No test item related effect on oestrus cycle of parental females was noted.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development.

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

The NOAEL for systemic toxicity of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for pup development and survival was considered to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted July 29, 2016

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"; deviations do not affect the validity of the study

Principles of method if other than guideline:

Due to technical reasons, the frozen plasma samples for the optional T4, T3 or TSH analyses were stored at -70 °C instead of -80 °C (nominal values), as indicated in the Study Plan.
Due to technical reasons, blood sampling from animal 3504 could not be performed; therefore, an additional animal (3508) from the same dose group was used for clinical chemistry and urine evaluation.
These deviations are considered to have no impact on the outcome of the study or on interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphinylidynetrimethanol

EC Number:

213-924-6

EC Name:

Phosphinylidynetrimethanol

Cas Number:

1067-12-5

Molecular formula:

C3H9O4P

IUPAC Name:

[bis(hydroxymethyl)phosphoryl]methanol

Test material form:

liquid

Details on test material:

- Appearance: light yellow to colourless ropy liquid
- Storage conditions at test facility: at 20 +/- 5 °C, in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat as a rodent is one of the standard strains for repeated dose toxicity studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Budapest, Hungary
- Age at study initiation: approximately 10 weeks
- Body weight range at study initiation: 316 to 421 g (males); 211 to 253 g (females)
- Fasting period before study: no
- Housing in T3H polycarbonate cages: The animals were group-housed, up to 3 animals of the same sex/cage, with the exception of the mating and gestation/delivery/lactation period, when they were paired or individually housed (with pups), respectively.
- Diet: standard laboratory rat diet, ad libitum [SM Rat/Mouse, Breeding & Maintenance, 15 mm, autoclavable; manufacturer: ssniff Spezialdiäten GmbH, Germany)
- Water: regularly monitored tap water from the municipal supply, as for human consumption, from drinking bottles, ad libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY:
The food is considered not to contain any contaminants that could affect the purpose or integrity of the study. The supplier provided an analytical certificate for the batch used, which is archived with the raw data.
Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary). The quality control results are retained in the archives of the laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 – 23 °C
- Humidity: 42 - 69 %
- Air changes: 15 to 20 per hour
- Photoperiod: 12 hours light (6:00 - 18:00) and 12 hours dark

IN-LIFE DATES: 2020-07-18 to 2020-09-24 (necropsy of last animals)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is the most feasible route to assess systemic exposure.

Vehicle:

water

Remarks:

distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test item was formulated in the vehicle (distilled water), as an aqueous solution at the appropriate concentrations. Formulations were prepared up to 8 days before use and were kept at room temperature until use. The formulation samples in the 1-220 mg/mL concentration range (using distilled water as vehicle) were proven as being stable for at least 21 days when stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: As determined in pre-tests, water was was selected as vehicle for this study.
- Concentration in vehicle: 1-220 mg/mL
- Amount of vehicle: 5 mL dose preparation/kg body weight
- Purity: 100% (distilled water, pharmaceutical grade Ph.Hg.VIII)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test item formulations for concentration and homogeneity was performed at the test site. Top, middle and bottom duplicate samples were taken from the test item formulations three times during the study (during the first and last weeks and approximately midway during the dosing period), one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected samples were stored at room temperature until shipment. Samples (one set) were shipped as soon as possible after collection for concentration and/or homogeneity measurement to the Principal Investigator (PI).
The formulation analyses were conducted under the control of the PI. Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration. Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) should be less than 10%.
The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirm that the formulations were homogeneous and stable from the time of preparation until completion of dosing.

Duration of treatment / exposure:

- Parental males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
- Parental females were dosed for 14 days pre-mating, for up to 6 days during mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition is complete) was defined as Day 0 post-partum. Females showing no evidence of pregnancy were sacrificed as practical (27-30 days after evidence of copulation).

Frequency of treatment:

Once daily (7 days / week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on the results of an oral gavage dose-range finding (DRF) study with THPO in the rat performed at the same test facility and using the same species and strain - where no clear test item related effects were seen up to 1000 mg/kg bw/day - doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Positive control:

No positive control required

Examinations

Observations and examinations performed and frequency:

- Clinical observations: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
General clinical observations were made once a day.
Detailed clinical observations were made on all animals outside the home cage in a standard arena at the start of the pre-exposure, prior to the first dosing (to allow for within-subject comparisons) and at least weekly thereafter, in the morning hours. Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc.), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

- Functional observation battery (FOB): Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 24-26; females on PPD 9-11). In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement.
Five males and five females per group were subjected to the functional observation battery, including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.
To measure the landing foot splay, the fore and hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots of the hind limbs were measured (the distance of the two forelimbs was not calculated as it was not deemed necessary by the Study Director).
Fore/hind grip strengths were measured using a grip strength meter (Nova ABF-50 Advanced with custom adapters). The rats were held appropriately such that the fore limbs allowed to grip the support bar and gently pulled back until they release the bar; the device measured the maximum grip strength. This was performed 3 times for each animal. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
Locomotor activity was recorded by placing the animals individually into an open-field for at least 1-hour observation time. Recording was made for a duration of at least 45 minutes, under dim-light and undisturbed conditions. The recorded video was processed and the calculated data was evaluated for distance travelled in 5 minute segments.

- Body weight measurements: Body weights of all parental animals were recorded with a precision of 1 g weekly during the pre-exposure period, on the first day of dosing (Day 0, prior to start of dosing), then afterwards at least weekly and prior to the scheduled necropsy (fasted).
Parental females were weighed on GD 0, 3, 7, 10, 14, 17, 20, and on PPD 0 (within 24 hours after parturition), 4, 7, 10, 13, and at termination (PPD 14, fasted). The body weight of the female animals measured on GD 3, 10 and 17 as well as PPD 7 and 10 were only additional measurements as aid for the calculation of accurate dosing volumes, and thus are not reported.

- Food consumption measurement: The determination of food consumption was performed weekly (on body weight measurement days). The remaining, non-consumed food was weighed with a precision of 1 g. Daily food consumption was calculated.

- Oestrus cycle monitoring: Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the dosing started. Vaginal smears were also checked daily from the beginning of the dosing period until evidence of mating (during the pre-mating and mating periods). Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

- Observation of the delivery process, offspring and nursing instinct: Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. Any evidence of abnormal deliveries were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy.
Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring were recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4 and PND 13, with accuracy of 0.01 g. All the litters were checked and recorded daily for the number of viable and dead pups, any abnormal behaviour or appearance of the pups was also recorded.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). The anogenital distance were also normalized to the pup size (the cube root of body weight). Number of nipples/areolae in male pups were recorded on PND 13.
One male and one female pup per litter (if possible) were selected for culling for blood sampling on PND 4. After that, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter. No pups were eliminated when litter size drooped below the culling target (10 pups/litter).
All remaining F1 offspring were terminated on PND 13. (In order to allow for overnight fasting of dams prior necropsy on PND 14, offspring were euthanized on PPD/PND 13, and the dams on PPD/PND 14.)

The following parameters were reported:
- Parental males:
Clinical observations and functional observation battery (FOB)
Body weight and body weight gain
Food consumption
Number of pairings
Number of fertile pairings
Number of infertile males
Male mating index
Male fertility index
Clinical pathology (including T4 hormone analysis)
Gross necropsy findings
Organ weights
Histopathology findings

- Parental females:
Clinical observations and functional observation battery (FOB)
Body weight and body weight gain
Food consumption
Oestrus cycle data (including number of females with normal and abnormal oestrous cycles)
Number of pairings
Number of pregnant females
Number of sperm positive, but non-pregnant females
Number of non-mated females
Female mating index
Female fertility index
Gestation index
Duration of pregnancy (in days)
Number of implantation sites / dams
Number of dams with live pups on Day 0, 4 and 13
Intrauterine mortality
Total offspring mortality (intra- and extrauterine mortality)
Clinical pathology
Gross necropsy findings
Organ weights
Histopathology findings

- Offspring:
Mean pup body weight (per pup within the group and per litter) on PND 0, 4 and 13
Mean pup body weight gain (per litter) between PND 0-4, 4-13 and 0-13
Number of live births per litter, and number of viable pups per litter on PND 0, 4 and 13
Survival index of pups on PND 0, 4 and 13
Sex ratio (for females) on PND 0, 4 and 13
T4 hormone analysis
Anogenital distance
Nipple retention
Thyroid glands weights

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see "Overall remarks")

HISTOPATHOLOGY: Yes (see "Overall remarks")

Statistics:

Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables. Frequency and percentage were calculated for categorical variables in Microsoft Excel.
Statistical analysis was performed for the continuous variables using an automated decision tree within the R software. The following decision tree was applied:
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in the study.

Mortality:

no mortality observed

Description (incidence):

No mortality was seen during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the body weight and body weight gain parameters of the test item treated animals in any of the sexes when compared to control data. The occasional statistically significances are considered incidental, not test item related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed on food consumption of the test item treated animals in any of the sexes when compared to control data. The statistically significant values in the male High dose group at the Day 14-21, 21-27 and 0-27 periods, and in the female Low dose group at the GD 14-20 period are considered incidental, toxicologically not relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed in the haematology parameters.
Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes were observed in the clinical chemistry parameters.
Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item related changes were observed in the urinalysis parameters.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of grip strength, landing foot splay or locomotor activity.
All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. Occasional statistical significance for an individual segment without a clear trend or dose response was not considered as test item related effect.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in the experimental animals.
There were no biologically relevant differences among groups in the weights of organs measured when compared to Controls in any sexes.
The absolute brain weight in the male High dose group and the relative (to the body weight) kidney and uterus weights in the female High dose group were statistically significantly different from the controls. These observed means were well within the historical control ranges in all cases, there were no clear dose responses and/or values were without any similar trend in the other sex of animals. Furthermore, there was no histopathological correlate for the weight changes. Thus, they were considered as having no toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related macroscopic changes were observed in the terminally euthanized animals.
The following findings were recorded at necropsy:
Dark red diffuse discoloration of the mandibular lymph nodes in 1/12 male Control, 2/12 male Low dose, red/dark red diffuse discoloration of the thymus in 1/12 male High dose and 1/12 female High dose animals.
Dark red single focal or multifocal discoloration of the glandular mucosa of the stomach was observed in 3/12 female Control, 2/11 female Low dose, 2/8 female Mid dose and 4/12 female High dose animals.
In one High dose male a cyst was observed on the right kidney, and also the left kidney was enlarged and the liver had multifocal dark red discoloration on all lobes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related microscopic changes were observed in the investigated reproductive and other organs of the animals.
The acute hemorrhage in the mandibular lymph node and thymus is considered as consequence of hypoxia, dyspnoe and circulatory disturbance developed during exsanguinations, without pathological significance.
Histological examination revealed in the stomach mucous membrane circumscribed congestion. No erosion, ulceration or inflammation was observed. It is considered to be caused by a mechanical effect, most probably related to the oral gavage of the animals.
The cyst in the kidney is considered as an individual disease.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The cyst in the kidney is considered as an individual disease.

Other effects:

no effects observed

Details on results:

- Mortality: No mortality was seen during the study.

- Clinical observation: No clinical signs were observed in the study.

- Body weight and body weight gain: No test item related changes were observed in the body weight and body weight gain parameters of the test item treated animals in any of the sexes when compared to control data. The occasional statistically significances are considered incidental, not test item-related.

- Food consumption: No test item related changes were observed on food consumption of the test item treated animals in any of the sexes when compared to control data. The statistically significant values in the male High dose group at the Day 14-21, 21-27 and 0-27 periods, and in the female Low dose group at the
GD 14-20 period are considered incidental, toxicologically not relevant.

- Neurological assessment: There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, landing foot splay or locomotor activity. All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. Occasional statistical significance for an individual segment without a clear trend or dose response was not considered as test item related effect.

- Haematology: No test item-related changes were observed in the haematology parameters. Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

- Clinical chemistry: No test item-related changes were observed in the clinical chemistry parameters. Statistically significant differences were observed in some cases, but there was no relationship with dose and/or all group means were well within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.

- Thyroid hormone analysis (adult): No effect of test item was observed in the thyroid hormone analysis. Compared to the control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males. Because of this, no further investigation was performed on the thyroid hormones (i.e. assessment of samples collected from the dams, assessment of other hormones, etc.).

- Urinalysis: No test item-related changes were observed in the urinalysis parameters.

- Organ weights: No treatment-related effects were observed in the experimental animals. There were no biologically relevant differences among groups in the weights of organs measured when compared to Controls in any sexes.The absolute brain weight in the male High dose group and the relative (to the body weight) kidney and uterus weights in the female High dose group were statistically significantly different from the controls. These observed means were well within the historical control ranges in all cases, there were no clear dose responses and/or values were without any similar trend in the other sex of animals. Furthermore, there was no histopathological correlate for the weight changes. Thus, they were considered as having no toxicological relevance.
 
- Pathology evaluation (macroscopic findings): No test item-related macroscopic changes were observed in the terminally euthanized animals. The following findings were recorded at necropsy: Dark red diffuse discoloration of the mandibular lymph nodes in 1/12 male Control, 2/12 male Low dose, red/dark red diffuse discoloration of the thymus in 1/12 male High dose and 1/12 female High dose animals; dark red single focal or multifocal discoloration of the glandular mucosa of the stomach was observed in 3/12 female Control, 2/12 female Low dose, 3/12 female Mid dose and 4/12 female High dose animals; in one High dose female a cyst was observed on the right kidney, and also the left kidney was enlarged and the liver had multifocal dark red discoloration on all lobes.

- Histopathology evaluation (microscopic findings): No test item-related microscopic changes were observed in the investigated reproductive and other organs of the animals. The acute hemorrhage in the mandibular lymph node and thymus is considered as consequence of hypoxia, dyspnoe and circulatory disturbance developed during exsanguinations, without pathological significance. Histological examination revealed in the stomach mucous membrane circumscribed congestion. No erosion, ulceration or inflammation was observed. It is considered to be caused by a mechanical effect, most probably related to the oral gavage of the animals. The cyst in the kidney is considered as an individual disease.

- Oestrus cycle evaluation in females / pre-exposure period: Each female selected for the study showed acceptable cycles before starting the dosing period. One animal had prolonged diestrus (presumably, it was the end of a pseudopregnancy) in the beginning of the pre-exposure period, but then the oestrus cyclicity became normal and thus the animal was not excluded from the study.
 
- Oestrus cycle evaluation in females / exposure period (pre-mating and mating periods): No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods. Prolonged diestrus (presumably pseudopregrancy) was recorded for one High dose female, but then the cycles of the animal became normal and the animal successfully mated and became pregnant. This is considered as being a normal finding, not being a test item related effect.

- Reproductive ability assessment and indices: There were no differences between the control and test item dosed groups with regard to reproductive ability, mating or gestation indices, that could be ascribed to the test item administration. Both the mating and fertility index was 100% in all groups (males and females). In the Low dose one female, and in the Mid dose 3 females did not get pregnant out of the 12 animals, which is considered normal. Also, no dose-dependency was observed. All females in the Control and the High dose groups became pregnant and delivered healthy litters. Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 6 days of pairing (cohabitation). The mean duration of mating was 2.83, 2.64, 2.30 and 2.42 days in the Control, Low, Mid and High dose groups, respectively.

- Evaluation of the pre-natal and gestation periods: There was no effect of treatment noted during the gestation period, parturition and postpartum period in any of dose groups. The mean duration of pregnancy was comparable in the Control and test item treated groups. As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted. The number of implantation sites was comparable to the control mean in all dose groups. There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values in any dose group.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

immunology

mortality

organ weights and organ / body weight ratios

urinalysis

other: serum/plasma biochemistry (migrated information); serum/plasma hormone analyses (migrated information); sperm measures (migrated information); other endocrine activity endpoints (migrated information)

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The study was performed acc. to OECD TG 422 under GLP, so the results can be considered reliable. No test-item related effects were noted throughout the study, so the no observed adverse effect level (NOAEL) for systemic, and reproductive and developmental toxicity is 1000 mg/kg/day under the conditions of this study. The test item is considered not to exhibit any toxicologically relevant effects up to the limit dose of 1000 mg/kg.

Executive summary:

The reproductive toxicity of Tris(hydroxymethyl)phosphine oxide (THPO) was investigated in a Reproduction/Development Toxicity Screening study following repeated daily administration by oral gavage to Wistar rats, conducted in accordance with the standardised guideline OECD 422, under GLP conditions.

The objective of the study was to screen for possible effects of the substance on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD13.

The test item was administered orally (by gavage) at 0, 100, 300 or 1000 mg/kg bw/day, corresponding to concentrations of 0, 20, 60 and 200 mg/mL at a 5 mL/kg bw dose volume to four dose groups of Wistar rats (n=12/sex/group) once a day.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessments, such as functional observation battery (FOB) including measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offsprings until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. A detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed.

In summary, daily administration of Tris(hydroxymethyl)phosphine oxide (THPO) by oral gavage to Hannover Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in mortality or any clinical signs.

The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.

At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.

No test item-related findings were noted in the clinical pathology parameters.

No test item related effect on oestrus cycle of parental females was noted.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development.

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

The NOAEL for systemic toxicity of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.