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Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 422 guideline GLP study (rat, oral gavage)

Link to relevant study records

Reference

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

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:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

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

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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

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.

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

See other information below.

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

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

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

See other information below.

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

 

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available data are considered suitable and reliable for REACH requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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. Details of these findings are presented in IUCLID Section 7.5, Repeated Dose Toxicity.
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.

Effects on developmental toxicity

Description of key information

OECD 414 guideline GLP study (rat, oral gavage, limit dose) - read across from structural analogue tris isotridecyl phosphite.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

NAME OF TEST MATERIAL (as stated in the study report): Trisisotridecylphosphite

SOURCE OF TEST MATERIAL
- Source of test material: Valtris Specialty Chemicals Ltd.
- Batch No. W057094
- Expiration date of the lot/batch: March 15, 2022
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in original container.
- Stability under test conditions: The stability of the test item in dose formulations was determined using a validated analytical method. The test item was found stable up to 24 hours under use conditions (i.e., room temperature). Dose formulations were prepared every day and used within 4 hours of preparation.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jai Research Foundation Animal Breeding Facility.
- Age at study initiation: At the initiation of the acclimatisation female rats were 11-13 weeks old.
- Weight at study initiation: 199.4 – 232.8 g at beginning of acclimatisation.
- Housing: female rats were housed individually, except during the mating period.
- Diet (e.g. ad libitum): standard rodent pellet diet (Certified Teklad Global 14% Protein Rodent Diet, Batch No 2014SC-090920MA,, procured from Envigo Laboratories, Inc., USA), ad libitum
- Water (e.g. ad libitum): filtered drinking water (filtered through Reverse Osmosis water filtration system) in polypropylene bottles, ad libitum.
- Acclimation period: 6 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 65 - 68
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours darkness, light hours were approximately 06.00 - 18.00 hours.

IN-LIFE DATES: Acclimatisation start March 05, 2021; Experiment Completion June 23, 2021.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared every day prior to the dosing and were used within 4 hours of preparation. Dose formulations were thoroughly mixed using a magnetic stirrer.

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

DOSE VOLUME: 4 mL/kg b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration and homogeneity of test item in samples of the control and dose formulations were analysed once prior to initiation of treatment and once during the treatment period. The samples were analysed at JRF using validated analytical method (JRF Study N° 228-2-13-18511).

Two sets of samples (4 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except control (only one aliquot). One set of samples was used for analyses and the other set of samples was stored at refrigerated. On each occasion, the mean concentration was determined and compared with the nominal value and the coefficient of variation calculated. The acceptance criteria was ±15% deviation from nominal value and %CV <10.

Details on mating procedure:

After the acclimatisation period, each female rat was cohabitated with an untreated male rat (1:1) until the requisite numbers of mated female rats (25/group) were obtained. Detection of mating was confirmed by evidence of a copulatory plug in the vagina or by sperm positive vaginal smear. After confirmation of mating, each the female rats were returned to an individual cage, assigned to a group and that day was designated as the day 0 of gestation (GD 0).

Duration of treatment / exposure:

Days 5 - 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

Day 20 of gestation

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 mated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The limit dose was chosen based on a testing requirement from ECHA following submission of a multi dose level OECD 414 guideline GLP study (JRF, 2018), which reported a NOAEL at the highest level tested (375 mg/kg/day).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the study period, visible clinical signs, such as changes in the skin, fur, eye, mucous membranes, as well as the behavioural pattern of the mated female rats, were recorded twice daily.

BODY WEIGHT: Yes
- Time schedule: on gestation days 0, 3, 5, 8, 11, 14, 17, and 20.

FOOD CONSUMPTION: Yes
- Time schedule: for the periods 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, 17–20, and 5-20 days of gestation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- All female rats were weighed, euthanised by carbon dioxide asphyxiation, dissected and examined macroscopically.

ORGAN WEIGHTS AND HISTOPATHOLOGY: Yes
- At the time of terminal sacrifice, the weight of the liver, kidney, brain, and thyroid gland was taken
after collection from all female rat and preserved in 10% neutral buffered formalin for histopathology.
Organ weights relative to terminal body weight and brain weight were calculated. Detailed histological examination of liver, kidney, and thyroid gland was performed in high and control dose groups. Brain was collected and preserved but histopathology was not performed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live fetuses: Yes
- Number of dead fetuses: Yes

Non-gravid uteri were further examined by immersing in 5% ammonium sulphide solution for confirming the non-pregnant status.

Blood sampling:

At the time of terminal sacrifice on GD 20, blood (approximately 3 mL) was collected from all-female
rats under anaesthesia (isoflurane) by orbital plexus puncture.

Serum thyroid hormones (T4, TSH, and T3) were analysed from all female rats at the terminal sacrifice.

Serum samples were also analysed for UDP Glucuronosyltransferase (UDPG) and clinical chemistry
parameters (alanine aminotransferase, aspartate aminotransferase, creatinine, total protein, albumin,
and urea) of liver and kidney.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [approximately half per litter]
- Skeletal examinations: Yes: [remaining half per litter]
- Head examinations: Yes: [approximately half per litter]
- Fetus sex (based on anogenital distance): Yes [all per litter]
- Fetuses body weight: Yes [all male fetuses per litter, all female fetuses per litter]

Statistics:

TYPE OF DATA:
Parametric: Body weight, body weight change, food consumption, corrected body weight,
absolute and relative uterine weight, organ weight, organ weight ratio, male sex ratio,
pre-and postnatal loss (%), live foetus (%), and resorption (%), ano-genital distance
Non-parametric: Mortality rate, pregnancy rate, foetal observation, litter size, number of corpora lutea, number of implants, number of a live foetus, number of pre-and postnatal losses,
number of resorptions

SIGNIFICANCE LEVEL:
Parametric: 5% and 1% level between control and treated group
Non-parametric: 5% level between control and treated group

Indices:

Pre-implantation loss (%)
Post-implantation loss (%)
Live fetuses (%)
Dead fetuses (%)
Male Sex ratio
Resorptions (%)

Historical control data:

No

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed during the experimental period in any dose group.

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was observed in any dose group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight and Day 20 corrected body weight of the pregnant female rats were comparable between the control and the test item treated group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the pregnant female rats was comparable between the control, and the test item treated group.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically, a significant increase in the serum ALT level was observed in the 1000 mg/kg b. wt./day
dose group. This alteration is considered an effect of the test item treatment but non-adverse in
nature in the absence of histopathological alterations in the liver.

Statistically, a significant increase in the serum UDP Glucuronosyltransferase (UDPG) level was
observed in the 1000 mg/kg b. wt./day dose group. This alteration reveals enzyme induction activity
of the test item and considered as an effect of the test item treatment but non-adverse in nature in the
absence of the histopathological alteration in the liver.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum TSH and T4 levels were comparable with that of the control group.

Statistically, a significant decrease in serum T3 level was observed in pregnant female rats belonging
to 1000 mg/kg b. wt./day dose group. This finding could be related to the induction of UDP
Glucuronosyltransferase (UDPG) seen in the liver, and associated increased clearance of the thyroid
hormones. In the absence of corresponding changes in TSH, and in the absence of histopathological alterations in thyroid, this finding is considered to be non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated group.

Statistically, a significant increase in the absolute (around 13% increase vs control) and relative
liver weights (vs terminal body weight and brain weight) were noted in the 1000 mg/kg b. wt./day
dose group. Similarly, statistically, a significant increase in the absolute (around 15% increase vs
control) and relative (vs brain weight) thyroid gland weights were observed in the 1000 mg/kg b. wt./day dose group. These effects were considered to be related to treatment but were considered nonadverse in nature in the absence of the treatment-related histopathological alterations in these organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External and internal (gross) examination of the terminally sacrificed female rats did not reveal any lesion of pathological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histological examination of liver, kidney, and thyroid gland did not reveal any treatment-related lesion
in rats of the control as well as the high dose group. The lesions observed, ectopic thymus and kidney pelvis dilation, were considered to be congenital/spontaneous as the lesions were present in both the treated and control group rats.

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

The mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions were comparable between the control, and test item treated group.

Dose descriptor:

NOEL

Remarks:

Maternal Developmental Toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: No treatment related effect observed at the limit dose of 1000 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

Maternal Systemic Toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: No adverse effects were observed at the limit dose of 1000 mg/kg/day.

Abnormalities:

no effects observed

Description (incidence and severity):

No adverse effects were observed at the limit dose of 1000 mg/kg/day.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of male, female and total fetuses (male + female) was comparable between the control and the test item treated groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, as well as the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions were comparable between the control, and test item treated group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male sex ratio was comparable between the control and the test item treated group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean count of male, female and total foetuses (male + female) was comparable between the
control group, and the test item treated group.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean anogenital distance of male and female foetuses was comparable
between the control and the test item treated group.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related external anomalies were observed.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No treatment-related increases in the incidence of skeletal malformations were observed. Findings were similar in the treated and control group, and at incidences within the range of historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related visceral anomalies were observed. Spontaneous findings in the treated and control group were within the historical control data range.

No treatment-related anomalies were observed in the head razor sections.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

No effects were observed in this study.

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No effects were observed at the limit dose of 1000 mg/kg/day.

Abnormalities:

no effects observed

Description (incidence and severity):

No effects were observed at the limit dose of 1000 mg/kg/day.

Developmental effects observed:

no

The following tabluar results and Appendices are provided in two separate attachments:

Table 2: Pregnancy Data Summary

Table 3: Clinical Observations Summary (Maternal)

Table 4: Body Weight Summary (Maternal)

Table 5: Body Weight Gain Summary (Maternal)

Table 6: Food Consumption Summary

Table 7: Prenatal Data Summary

Table 8: Foetal Data Summary

Table 9: Foetal Gross External Observation Summary

Table 10: Foetal Visceral Observations Summary

Table 11: Foetal Head Razor Section Observation Summary

Table 12: Foetal Skeletal Observation Summary

Appendix 11: Pathology Report

Appendix 12: Thyroid Hormone Analysis Report (TSH)

Appendix 13: Thyroid Hormone Analysis Report (T3 and T4)

Appendix 14: UDPG Analysis Report

Conclusions:

In this OECD 414 guideline GLP limit study of trisisotridecyl phosphite, no adverse maternal systemic effects were observed at the limit dose of 1000 mg/kg bw/day, and no treatment-related effects were observed on maternal reproduction or fetal development. Therefore, the NOAEL for maternal systemic toxicity is considered to be 1000 mg/kg bw/day. This dose in considered to be a NOEL for maternal and fetal developmental toxicity.

Executive summary:

An enhanced OECD 414 guideline GLP limit study was conducted on tris isotridecyl phosphite. Groups of 25 female Wistar rats were treated by oral gavage on gestation days (GD) 5 to 19, by at  dose levels of 0 (corn oil vehicle), or 1000 mg/kg b. wt./day. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period.

All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised, and the number of corpora lutea counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. At the time of terminal sacrifice, the weight of the thyroid gland, liver, kidney, and brain was recorded from all-female rats and preserved for histopathology. Detailed histological examination of the liver, kidney, and thyroid gland was performed in control and high dose group female rats. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid-stimulating hormone) were analysed from all pregnant female rats during the terminal sacrifice. UDP Glucuronosyltransferase (UDPG) and clinical chemistry parameters (alanine aminotransferase, aspartate aminotransferase, creatinine, total protein, albumin, and urea) of liver and kidney were also analysed from all pregnant female rats.  The maternal liver evaluations were included to aid interpretation of thyroid findings. Following appropriate fixation, foetuses were examined for visceral abnormalities, including razor sectioning of the head and skeletal abnormalities.

No treatment-related effects were seen on maternal reproduction parameters, or on fetal development.

Treatment-related effects were limited to liver and thyroid findings in maternal animals.  Nonadverse effects seen in the 1000 mg/kg b. wt./day dose group animals included:

- Statistically significant increase in the serum ALT level vs controls.

- Statistically significant increase in the serum UDP Glucuronosyltransferase (UDPG) vs controls. This finding indicates enzyme induction activity in the liver and and has been  associated with increased clearance of the thyroid hormones.

 - Statistically significant decrease in serum T3 level vs controls. This finding could be related to the induction of UDP Glucuronosyltransferase (UDPG) seen in the liver.

- Statistically significant increase in the absolute and relative liver and thyroid weights vs controls.

Because no treatment-related histopathological alterations were seen in the liver or thyroid, these findings were considered to be non-adverse in nature.

Based on these results, the NOAEL for maternal systemic toxicity is considered to be 1000 mg/kg bw/day. This dose in considered to be a NOEL for maternal reproduction and fetal developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available data are considered suitable and reliable for REACH requirements.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Data on a closely related structural analogue, tris isotridecyl phosphite (TiTDP), has been selected to represent the prenatal developmental toxicity of tris 2-propylheptyl phosphite (T2PHP). Based on similar toxicity on other mammalian endpoints it appears appropriate to use this TiTDP data to read-across to T2PHP. In addition, the OECD- accepted category approach for the C10 and C13 isoalkyl alcohols, hydrolysis products of T2PHP and TiTDP, respectively, further supports this read-across approach.

An enhanced OECD 414 guideline GLP limit study was conducted on tris isotridecyl phosphite (TiTDP). Groups of 25 female Wistar rats were treated by oral gavage on gestation days (GD) 5 to 19, by at dose levels of 0 (corn oil vehicle), or 1000 mg/kg b. wt./day. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period.

All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised, and the number of corpora lutea counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. At the time of terminal sacrifice, the weight of the thyroid gland, liver, kidney, and brain was recorded from all-female rats and preserved for histopathology. Detailed histological examination of the liver, kidney, and thyroid gland was performed in control and high dose group female rats. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid-stimulating hormone) were analysed from all pregnant female rats during the terminal sacrifice. UDP Glucuronosyltransferase (UDPG) and clinical chemistry parameters (alanine aminotransferase, aspartate aminotransferase, creatinine, total protein, albumin, and urea) of liver and kidney were also analysed from all pregnant female rats.  The maternal liver evaluations were included to aid interpretation of thyroid findings. Following appropriate fixation, foetuses were examined for visceral abnormalities, including razor sectioning of the head and skeletal abnormalities.

No treatment-related effects were seen on maternal reproduction parameters, or on fetal development.

Treatment-related effects were limited to liver and thyroid findings in maternal animals and considered to be non-adverse in nature given the absence of histopathological alterations.  Statistically significant increases in the serum UDP Glucuronosyltransferase (UDPG) were seen in the treated maternal animals vs controls. This finding, which indicates enzyme induction activity in the liver has been associated with increased clearance of thyroid hormones in rodents.  This rodent-specific effect has been reviewed and is considered of insufficient concern for classification (see ECBI/22/98 Add1, EU Commission Meeting of the Commission Working Group on C&L of Dangerous Substances ECBI/27/98 Rev.2).

Based on these results, the NOAEL for maternal systemic toxicity of TiTDP is considered to be 1000 mg/kg bw/day. This dose in considered to be a NOEL for maternal reproduction and fetal developmental toxicity.

 

In a OECD 422 guideline GLP study of tris 2-propylheptyl phosphite, no treatment-related effects were observed on pup survival, growth or development at the highest dose tested, 450 mg/kg bw/day.

Justification for classification or non-classification

In an OECD 422 guideline GLP rat study of tris 2-propylheptyl phosphite by oral gavage at doses up to 450 mg/kg bw/day, 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. The fertility and reproductive NOAEL for parental animals and the developmental NOAEL for the F1 pups was 450 mg/kg/day.

In an OECD 414 guideline GLP rat limit study conducted on the closely related structural analogue tris isotridecyl phosphite at the limit dose of 1000 mg/kg bw/day, no treatment-related effects were seen on maternal reproduction parameters, or on fetal development.

The available data are considered suitable and reliable for REACH requirements.

Based on these data, the substance is not considered to be classified for reproductive toxicity under Regulation (EC) 1272/2008.