[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2022

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:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-propylheptyl) phosphite

Molecular formula:

C30H63O3P

IUPAC Name:

Tris(2-propylheptyl) phosphite

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Valtris Specialty Chemicals Ltd, lot/batch number W051290.
- Purity, including information on contaminants, isomers, etc.: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in original container.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test material concentrations were shown by testing to be stable in the corn oil vehicle after 24 hours. Dose formulations were prepared and used daily.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal breeding facility at Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: [yes/no] yes
- Age at study initiation: (P) 11-12 wks
- Mean Group Weight at study initiation: (P) Males: 388-392 g; Females: 246-249 g
- Housing: Rats were housed in groups of 2 or 3 rats/sex/cage during premating period. During the mating period, rats were housed in groups of 2 rats/cage (one male plus one female) and mated female rat was caged individually. Enrichment material was provided to all rats. Nesting material was provided at near parturition (from gestation day 14). During the study, rats were housed in solid floor polypropylene rat cages. Each cage was fitted with a stainless-steel top grille having provision for a polypropylene water bottle with a stainless steel drinking nozzle. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on a 5 tier racks. Cages and enrichment material were changed minimum twice a week. Cages were arranged in such a way that possible effects due to cage placement were minimised.
- Diet (e.g. ad libitum): Rats were fed ad libitum with standard rodent diet (Teklad Certified Global 16% Protein Rodent Maintenance Diet, Batch 2016SC-120519MA procured from Envigo Laboratories, Inc., USA).
- Water (e.g. ad libitum): Rats were provided reverse osmosis hi-tech sweet water (RO) water ad libitum (filtered through RO water filtration system) in polypropylene bottles.
- Acclimation period: Seven days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 degrees
- Humidity (%): 65-68
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: April 9, 2020 To: July 10, 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was mixed with the appropriate volume of corn oil vehicle to prepare dose formulations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and during the dosing. Dose formulations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Two weeks maximum.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- Acyclic female rat was assumed as mated after the completion of two-weeks mating period.
- Re-mating of cyclic female with proven males of the same group was performed.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity of the test item in the vehicle were analyzed once before initiation of treatment and twice during the treatment period using a validated GC-FPD method. The mean percent recovery obtained for the test doses were within the acceptance level of ±15% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 10%, indicating that the test doses were homogeneously prepared.

Duration of treatment / exposure:

Dosing of both sexes was initiated 2 weeks prior to the mating and continued during the mating period. After mating, the male rats were further dosed up to and including the day before scheduled sacrifice on study Day 32. Female rats were dosed during pregnancy and up to post-partum day 14.

Rats belonging to recovery groups were kept for 15 days after the first scheduled sacrifice of dams, without treatment.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 13-14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day

No. of animals per sex per dose:

Main control and treated groups: 15 males and 15 females
Recovery groups (control and high dose only): 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a 28-day dose range finding study (JRF Study N° 410-1-04-24390) where significant effects on organ weights were observed at 500 and 1000 mg/kg b. wt./day.
- Fasting period before blood sampling for clinical biochemistry: Food was withheld overnight.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during treatment period; once daily during recovery period.
- Cage side observations: mortality, morbidity, visible clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Neurobehavioral: prior to initiation of treatment and weekly thereafter.
- Functional Observational Battery: 5 rats/sex/group randomly selected near the end of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - On the first day of dosing and weekly thereafter. Females - On the first day of dosing and at weekly intervals during the pre-mating and the mating periods. During the gestation period, female rats were weighed on gestation days 0, 7, 14, 20, and 25. During the lactation period, female rats were weighed within 24 hours of parturition (day ‘0’ post-partum/lactation day), and on post-partum days 4, 7, and 14.

FOOD CONSUMPTION: Yes
- Time schedule: The food consumption was determined by differentiating the weight of food input and leftover.
Food weights of male rats were determined weekly during the pre-mating and post-mating periods.
In female rats, during the pre-mating period, food weights were recorded at weekly intervals. During the gestation period, food weights were measured on days 0, 7, 14, and 20. During the lactation period, food weights were measured on days 0, 4, 7, and 14.
Food consumption was not measured during the mating period.
Food weights of male and female rats belonging to recovery groups were determined weekly throughout treatment and recovery periods.

WATER CONSUMPTION: No

CLINICAL PATHOLOGY (hematology, clinical chemistry, thyroid hormones, urine): Yes
- Time schedule: At terminal sacrifice, blood was collected from all surviving rats under anaesthesia (isoflurane) by orbital plexus puncture. Rats were deprived of food overnight (allowed water ad libitum) prior to blood collection. Blood samples were collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry, and thyroid hormone (T3, T4, TSH) analysis (in vials without anticoagulant).

At the time of terminal sacrifice, urine samples were collected overnight from five rats (adult)/sex/group in graduated collecting tubes.

Oestrous cyclicity (parental animals):

Oestrous cycle length and pattern were evaluated by vaginal smears observation of individual female rats during the pre-treatment period of two weeks. Vaginal smear was monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant rat, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- The size of each F1 litter was adjusted by removing extra pups by random selection to yield, as close as possible, four male and four female pups per litter. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (e.g., six males and two females) was performed. Adjustments were not performed for litters of eight pups or less.

PARAMETERS EXAMINED
- The following parameters were examined in offspring: Each litter was examined as soon as possible after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Each pup was observed for the presence of milk in the stomach on PND 0 to ensure nursing care. AGD of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13.

PUP BODY WEIGHT
- Individual pup body weight was recorded on PND 0, 4, 7, and 14.

THYROID HORMONES
- Blood was collected through decapitation for serum thyroid hormone analysis from two surplus pups/litter (wherever available) on postnatal day 4 (for T4), and two pups/litter on the day of terminal sacrifice i.e., PND 14 (for T3, T4, TSH). Pup blood was pooled by litter.

GROSS EXAMINATION OF DEAD PUPS:
Pups which died during lactation were weighed and subjected to a post-mortem examination. Pups found dead on the day of littering were examined for possible defects and cause of death and discarded in the absence of gross findings.

Postmortem examinations (parental animals):

SACRIFICE
- Main male animals: Males of the main groups were treated up to the day 32 and sacrificed on day 33.
- Main female animals: Female rats were sacrificed on LD 15. Females that did not mate were sacrificed by 25 days after the last day of mating period. Females that did not deliver by day 25 post-coitum were sacrificed at that time.
- Recovery animals: Male and female rats belonging to recovery groups were sacrificed 15 days after the first scheduled sacrifice of the main study dams.

GROSS NECROPSY
- Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out the blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities. Special attention was paid to organs of the reproductive system.

The uteri of all cohabited female rats were examined for the presence and number of implantation sites.

ORGAN WEIGHTS
- The following organs / tissues: testes, epididymides, Levator ani plus bulbocavernosus muscle complex, Cowper's glands, glans penis, ovaries, thyroid, uterus with oviducts and cervix, adrenals, liver, kidneys, thymus, spleen, brain, heart.

HISTOPATHOLOGY / ORGAN WEIGHTS
- A detailed histopathological examination of 31 preserved organs including gross lesions was performed in high and control dose group rats.

Detailed testicular histopathological examination, paraffin embedding and transverse sections of 4-5 µm thickness) was conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation included identification of treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. Periodic Acid Schiff (PAS), haematoxylin, and eosin staining was used for examination of the testes, while haematoxylin and eosin were used for epididymides and ovaries.

Histopathological examination of the ovary was carried out to detect treatment-related effects such as qualitative depletion/increase of primordial, secondary, antral, graffian follicles population, persistence and increased/decreased corpus luteum, ovarian degeneration/atrophy and stromal cell proliferation.

In addition, all gross lesion, as well as liver and thyroid were processed from all lower dose groups and all recovery group and examined microscopically.

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed on postnatal day 14.

GROSS NECROPSY
- At the time of sacrifice or death during the study, all pups were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Pups which were found dead on PND 0 were subjected to gross examination and portion of lung was immersed in water for confirmation of live and dead status at the time of delivery (stillbirth or dead). Pups which were found dead during lactation were observed for the presence of milk band. Pups without gross lesions were discarded after the examination.

Statistics:

Non-pregnant female rats were excluded from statistical analysis.
Data such as body weight, body weight gain, food consumption, hind limb foot splay, grip strength, organ weight, organ weight ratio, % pre-natal loss, % post-natal loss, and litter parameters (pups body weight and pups body weight gain) were subjected to Shapiro-Wilk’s test for checking normality wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data did not meet the normality, data were transformed to check the normality again. When log transfer data did not meet the normality, Kruskal-Wallis test or Mann-Whitney test were performed to calculate significance. When the data did not meet the homogeneity of variance, statistical analysis was extended following the decision tree (Gad, S.C., 2007). AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
Countable data {viz., litter size, number of implants, pre-coital interval, duration of gestation, number of the oestrous cycles, urination count, defecation count, rearing count, motor activity (total, fine and ambulatory)} were subjected to non-parametric test either Kruskal-Wallis test or Mann-Whitney test.
Non-parametric data such as gestation index, parturition index, pregnancy rate, survival index, mortality index, live birth index and fertility index were analysed using a Chi-Square test.

Reproductive indices:

Mating indices
Fertility indices
Gestation index
Parturition index
Male sex ratio

Offspring viability indices:

Pre- and post-natal loss
Live birth index
Pup survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation (mild) was observed approximately 3 to 5 minutes after dosing in male at 450 and in female rats at 150 and 450 mg/kg b. wt./day dose groups and persisted for approximately 40 to 45 minutes. This finding is considered a response to the dose solution and toxicologically non-adverse in nature.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Serum TSH levels of male rats treated at 150 and 450 mg/kg b. wt./day were statistically significantly increased when compared with that of the control group. Statistically significant decrease was noted in serum T3 levels at 150 and 450 mg/kg b. wt./day and T4 at 450 mg/kg b. wt./day when compared with those of the control group.

Serum TSH, T3 and T4 level of male rats at 50 mg/kg b. wt./day were comparable with that of the control group.

TSH, T3 and T4 levels of female rats for all the dose levels were comparable with those of the control group.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed in liver and thyroid.

Microscopic examination of liver revealed centrilobular hypertrophy in two mid-dose male animals and diffuse hypertrophy in high-dose animals of both sexes, as well as in female high-dose recovery animals. The lesions were well correlated with increased liver weight in main and recovery groups. Further these lesions were minimal to mild in nature. These changes might be due to induction (upregulation) of xenobiotic-biotransforming enzymes and transporters, an adaptive process that augments xenobiotic elimination during periods of high xenobiotic exposure (Curtis, 2008).

The hepatocellular hypertrophy ceased completely in recovery male animals, whereas in female animals the effect persisted with lesser incidence, which suggests additional recovery period could have resulted in complete recovery. Moreover, the test item did not result in any treatment related alteration in clinical chemistry parameters related to liver function (ALT, AST, ALP, GGT and total bilirubin). Therefore, this effect could be considered as an adaptive response.

Microscopic examination of thyroid revealed minimal to mild follicular cell hypertrophy in most animals of the mid-dose and high-dose groups, and in two males and two females in the low-dose group. This effect persisted in male and female animals of the high-dose recovery group.

Thyroid gland follicular cell hypertrophy and hepatocellular hypertrophy are often seen concomitantly in rats. An increase in liver weight, associated with liver enzyme induction and seen as centrilobular hypertrophy, is commonly observed along with thyroid follicular cell hypertrophy, and is a physiological response (Catherine and Philip, 2015).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum UDPG transferase levels of the high dose group were comparable to the control group levels, based on ELISA kit analysis of remnant serum samples stored at -70 +/- 10 degrees C.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

See other information below.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Reproductive Effects

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects seen at the highest dose tested

2

Dose descriptor:

LOEL

Remarks:

Systemic effects

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

other: thyroid hormones

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Mortality and survival index of male, female and composite of male and female pups, belonging to the 50, 150 and 450 mg/kg b. wt./day dose groups, were comparable with that of the control group.

Body weight and weight changes:

no effects observed

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:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

See other information below.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental effects

Generation:

F1

Effect level:

450 mg/kg bw/day

Based on:

other: Maternal dose

Sex:

male/female

Remarks on result:

other: no effects observed at the highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

The following tabular results are provided in an attached file.

Table 9: Summary of Litter Size and Male Sex Ratio

Table 10: Summary of Pup Body Weight and Ano-Genital Distance

Table 11: Summary of Pup Body Weight Gain

Table 12: Summary of Fertility, Reproduction and Development

Additional information for parental systemic effects are provided in IUCLID Section 7.5, Repeated dose toxicity.

 

Applicant's summary and conclusion

Conclusions:

- A clear systemic No Observed Adverse Effect Level (NOAEL) in parental rats was not established, due to uncertainty about the association between liver and thyroid findings and their significance (adverse or adaptive). The Lowest Effect Level (LOEL) was considered to be 150 mg/kg b. wt./day based on minimal to mild thyroid follicular cell hypertrophy (males and females) and thyroid hormone changes (males only).
- The reproductive and fertility NOAEL in parental rats was found to be 450 mg/kg b. wt./day as no treatment-related effect was observed on reproduction and fertility endpoints.
- The developmental NOAEL in F1 pups was found to be 450 mg/kg b. wt./day as no treatment-related effect was observed on growth, survival, and external abnormality of pups.

Executive summary:

An OECD 422 guideline GLP study was conducted of tris 2-propylheptyl phosphite.  Groups of 15 male and 15 female Wistar rats were administered the test substance by oral gavage at doses of 0 (corn oil vehicle only), 50, 150 or 450 mg/kg/day.  Doses were administered daily for two weeks prior to mating, and during a two-week mating period. Following mating, males continued on treatment until sacrifice on study day 33.  Females continued on treatment through gestation, parturition, and until sacrifice on lactation day 14.  Two separate groups of 5 male and 5 female animals were treated at the high dose or with corn oil for seven weeks (without mating) and then held for an additional 2 weeks to evalute the reversibility of any effects. 

Parental animals were evaluated for systemic effects, fertility and reproduction. Litters were culled on Day 4 of lactation and development of the remaining pups evaluated until study termination on lactation day 14.

Treatment-related systemic effects in parental animals were limited to the liver and thyroid. 

Minimal to mild hepatocellular hypertrophy was seen in 150 and 450 mg/kg b.wt./day males with corresponding liver weight increases at 450 mg/kg b.wt./day.  Clinical chemistry parameters related to liver function (ALT, AST, ALP, GGT, total bilirubin, UDPG transferase) were similar to control results, therefore, the liver hypertrophy could be considered as an adaptive response.  Similar liver effects were seen in the 450 mg/kg b.wt.\day females.

Mild thyroid follicular cell hypertrophy was seen in 150 and 450 mg/kg b.wt./day males along with corresponding changes in thyroid hormone levels (decreased T3/T4 and increased TSH), and increased thyroid weight at 450 mg/kg b.wt./day.  Similar hypertrophy was seen in 150 and 450 mg/kg b.wt./day females, but without corresponding changes in hormone levels or thyroid weight.

Thyroid gland follicular cell hypertrophy is often seen concomitantly in rats with hepatocellular hypertrophy (adaptive liver changes). In this study, there was dose concordance between the liver and thyroid effects in males, but not in females. 

After the 14-day recovery period, hepatocellular hypertrophy resolved in the males, but not in the females.  Thyroid follicular cell hypertrophy persisted in both males and females.

An increase in liver weight, associated with liver enzyme induction and seen as centrilobular hypertrophy, is commonly observed along with thyroid follicular cell hypertrophy, and is a physiological response (Catherine and Philip, 2015).  However, given the lack of dose concordance between liver and thyroid effects in females, similar UDPG levels measured in contol and high-dose animals, and the persistence of thyroid effects in male and female recovery animals, association of the observed thyroid effects with adaptive changes in the liver cannot be clearly established. Therefore a clear systemic NOAEL in parental rats was not established, due to uncertainty about the association between liver and thyroid findings and their significance (adverse or adaptive). The Lowest Systemic Effect Level (LOEL) in parental animals was considered to be 150 mg/kg b. wt./day based on minimal to mild thyroid follicular cell hypertrophy (males and females) and thyroid hormone changes (males only).

No treatment-related effects were observed on fertility or reproduction of the parental animals.  Similarly, no treatment-related effects were observed on pup survival, growth or development. Accordingly, the fertility and reproductive NOAEL for parental animals and the developmental NOAEL for the F1 pups was found to be 450 mg/kg/day.