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Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Triphenyl phosphite
EC Number:
202-908-4
EC Name:
Triphenyl phosphite
Cas Number:
101-02-0
Molecular formula:
C18H15O3P
IUPAC Name:
triphenyl phosphite
impurity 1
Chemical structure
Reference substance name:
Phenol
EC Number:
203-632-7
EC Name:
Phenol
Cas Number:
108-95-2
Molecular formula:
C6H6O
IUPAC Name:
phenol
Test material form:
liquid
Specific details on test material used for the study:
Manufactured by UPL Ltd. (India)
Batch #: JQ05TPIB87

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in original containers
- Stability: The stability of the test item in the vehicle was established in JRF Study number 228-2-13-24482. Based on results, it was observed that the test item in the vehicle was stable up to 24 hours.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Details on test animals or test system and environmental conditions:
Healthy young adult rats (Rattus norvegicus) of Wistar strain (RccHan:WIST) were obtained from the Animal Breeding Facility, Jai Research Foundation and used for the experiment. Female rats (11-13 weeks old) were nulliparous and non-pregnant at the initiation of the acclimatisation. Animals were acclimated in the experimental room for a period of 7 days prior to the cohabitation for mating. After getting 100 sperm positive females (25 mated females/group), the remaining rats were returned to the Animal Breeding Facility, JRF or discarded without any further examination.

Rats were maintained in an environment-controlled room at a temperature of 22 ± 3 °C and relative humidity of 30 to 70 percent. The photoperiod was 12 hours fluorescent light and 12 hours darkness, light hours being 06.00 - 18.00 hours and air changes with filtered fresh air will be minimum of 15 per hour. The mean light intensity in an animal room will be maintained between 130 to 325 Lux.

Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male plus one female). Mated female rats were housed individually. Enrichment material (wooden chew block) was provided to all rats. During the study, rats were housed in solid floor polypropylene rat cages (size: 427 x 287 x 198 mm). Each cage was fitted with a stainless-steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks. The bottom of the cages was layered with clean sterilised rice husk as the bedding. Cages with bedding material and water bottles were changed twice a week.

The rats were fed ad libitum with standard rodent pelleted diet (Certified Teklad Global 14% Protein Rodent Diet, Batch No 2014SC-120519MA, procured from Envigo, Inc., USA) with an unlimited supply of drinking water in polypropylene bottles. Drinking water was filtered through the Reverse Osmosis water filtration system.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dose formulations were prepared every day prior to the dosing and thoroughly mixed using magnetic stirrer in order to make a homogeneous preparation. Dose-volume was 4 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two sets of samples were collected by sampling three aliquots (top, middle, and bottom layers) from each concentration except the vehicle control (only one aliquot from the middle layer). Sampling was done before the start of treatment and once during the treatment period for dose formulation analyses.
Details on mating procedure:
After completion of the acclimatisation period, each female rat was cohabitated with an untreated male rat until the requisite numbers of mated females were obtained. Detection of mating was confirmed by the evidence of a copulatory plug in the vagina or by vaginal lavage for sperm. After confirmation of mating, each female rat was returned to an individual cage, assigned to a group, and that day was designated as day 0 of gestation (GD 0).
Duration of treatment / exposure:
Gestation Days 5 to 19
Frequency of treatment:
The test item was administered daily to the three groups of pregnant female rats while the control group received the vehicle only.
Duration of test:
Acclimation: 7 days
Mating: up to 2 weeks
Gestation: 20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
110 mg/kg bw/day
No. of animals per sex per dose:
25 pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on results obtained in a dose range-finding study (JRF Study N° 461-1-04-24483)

Examinations

Maternal examinations:
Mortality and Morbidity: Rats were observed twice daily for mortality and morbidity. Females who were found dead/moribund during the study were weighed and subjected to the post-mortem examination.

Clinical Signs: 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: Body weights for the mated female rats were recorded individually on days 0, 3, 5, 8, 11, 14, 17, and 20 of gestation.

Food Consumption: A fixed amount of pelleted rat food was presented to each mated female rat, and the food remaining at the end of the specified interval was recorded. The food consumption was determined for the following intervals: Days 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, 17-20, and 5-20.

Blood Collection: At the time of terminal sacrifice, blood (approximately 1 mL) was collected from all surviving female rats under anaesthesia (isoflurane) by orbital plexus puncture.

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

Necropsy: On day 20 of gestation, all surviving female rats were weighed, euthanised by an overdose of carbon dioxide, dissected and examined macroscopically.

At the time of terminal sacrifice, the weight of the brain and thyroid gland was recorded from all surviving and two moribund sacrificed female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in high and control dose group rats.
Ovaries and uterine content:
The non-gravid uteri were further examined by staining with 5% ammonium sulphide to confirm non-pregnant status.
Ovaries and gravid uteri including the cervix, were removed and examined immediately for following parameters listed below. Ovaries, foetuses, and non-gravid uteri were preserved and identified.

Maternal Parameters:
Gravid uterine including the cervix weight (g)
Number of corpora lutea (CL)
Number of implantation sites (IS)
Number of live foetuses (LF)
Number of dead foetuses (DF)
Number of early resorptions (ER)
Number of late resorptions (LR)
Number of total resorptions (TR)

Fetal examinations:
Foetal Observations:
The foetuses of all surviving rats were examined for gross external anomalies and findings were recorded.

Soft Tissue Evaluation:
Approximately 50% of the foetuses from each surviving litter were subjected to the detailed visceral evaluation by microdissection. The cephalic evaluation was done by head razor sectioning after fixation in acetoalcohol formalin solution (i.e., Davidson’s fixative). The procedure for evaluation of soft tissue was according to Staple’s technique.

Skeletal Evaluation
The remaining 50% of the foetuses from each surviving litter were subjected to the detailed skeletal evaluation after processing and staining with alizarin red. The foetuses were preserved in isopropyl alcohol and glycerin solution. The skeletal evaluation was performed according to Staple’s and Schnell method.

Other Foetal Parameters:
Number of male foetuses
Number of female foetuses
Body weight (g) of male foetuses
Body weight (g) of female foetuses
Foetus sex (based on anogenital distance)
Ano-genital distance (mm)

Statistics:
All raw data were processed to determine group means and standard deviations with statistical significance between the control and treatment groups using validated statistical software.
The parametric data (body weight, body weight change, food consumption, hormones (T3, T4, and TSH) results, and organ weight, organ weight ratio, male ratio, percent pre-implantation loss, percent post-implantation loss, percent live foetus, and percent resorption) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data do not meet the homogeneity of variance, statistical analysis was extended following a decision tree (Gad, S.C., 2007).
The non-parametric data (mortality rate, pregnancy rate, foetal observations etc.) were analysed using the Chi-square test.
Count data (viz., foetal count, number of corpora lutea, number of implants, number of live foetuses, number of dead foetuses, number of early, late, and total resorptions) were subjected to non-parametric Kruskal-Wallis test.
AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
Non-pregnant, found dead and moribund rats were not subjected to statistical analysis.
Indices:
Parameters Calculated:
Number and percent of pre-implantation loss
Number and percent post-implantation loss
Live foetus (%)
Dead foetus (%)
Male sex ratio
Corrected body weight
Percent of resorptions

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs, such as abnormal gait, piloerection, weakness, chromodacryorrhea, diarrhoea, rough hair coat, and ataxia were observed at 110 mg/kg b. wt./day. There were no clinical signs observed in the 0, 15 and 40 mg/kg b. wt./day dose groups.
Description (incidence and severity):
Study results, including fetal examinations, have yet to be provided by the laboratory. Expected in December 2020.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of two female rats were found dead (rat No 84 and 91 on GD 20 and 17, respectively) at 110 mg/kg b. wt./day dose group.
In addition, a total of four female rats in the 110 mg/kg b. wt./day dose group were observed moribund (rat No 88, 89, 93, and 100 on GD 20, 20, 19, and 16, respectively). All these females were humanely sacrificed. There were no unscheduled deaths or moribund females in the 0, 15 and 40 mg/kg b. wt./day dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly lower mean body weight (on GD 17 and 20) and the corrected body weight (on GD 20) were observed in pregnant rats of the 110 mg/kg b. wt./day dose group when compared with that of the vehicle control group. The lower body weight was corresponded to decrease food consumption at 110 mg/kg b. wt./day dose group. This effect was considered as a treatment-related adverse effect of the test item.
Significant changes in body weight (on GD 0 and 3) of pregnant rats of the 15 and 40 mg/kg b. wt./day dose groups were considered as incidental without any toxicological relevance because the treatment was initiated on GD 5.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in mean food consumption (during GD 5-8, 11-14, 14-17, 17-20, and 5-20) were observed in pregnant rats of the 110 mg/kg b. wt./day dose group when compared with that of the vehicle control group. This effect was related to treatment and considered as an adverse effect of the test item.
Significant increase in food consumption (during GD 3-5) of pregnant rats of the 15 and 40 mg/kg b. wt./day dose groups were considered as incidental without any toxicological relevance because the treatment was initiated on GD 5.
Food efficiency:
not examined
Description (incidence and severity):
.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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):
Absolute and relative (to brain) weights of thyroid gland were comparable to control values. Significantly lower brain weights in 110 mg/kg b. wt/day dose group are considered to be a reflection of lower body weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal (gross) examination of moribund, found dead, and terminally sacrificed female rats did not reveal any lesion of pathological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Thyroid: Examination of the thyroid gland of female rats at the 110 mg/kg b. wt./day dose group did not reveal any histopathological changes.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum T3 and T4 levels of GD 20 female rats, belonging to the 110 mg/kg b. wt./day, were statistically significantly decreased when compared with that of the vehicle control group. However, serum TSH level of GD 20 female rats were comparable. This effect was considered an indirect effect of the test item related to overt maternal toxicity. Thyroid hormone levels in the 15 and 40 mg/kg b. wt/day dose groups were comparable to control values.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No aborted fetuses were observed in the study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
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 vehicle control and test item treated groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See description above.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See description above.
Dead fetuses:
no effects observed
Description (incidence and severity):
See description above.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
All animals surviving the study had litter data taken on gestation Day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Control Group: 92%
15 mg/kg b. wt/day Group: 100%
40 mg/kg b. wt/day Group: 96%
110 mg/kg b. wt/day Group: 92%

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
110 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly lower mean body weights of male, female and total foetuses (male + female) were noted at 110 mg/kg b. wt./day when compared with that of the vehicle control group. These lower foetus body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the lower maternal body weight. Fetal body weights in the 15 and 40 mg/kg b. wt/day dose groups were comparable to control values.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Mean numbers were comparable between the control and treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratios were comparable between the control and treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean numbers were comparable between the control and treated groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related external anomalies were observed in foetuses of the treatment groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were several incidences of statistically significant differences in the skeletal variation in the 110 mg/kg b. wt./day dose group. These findings are considered secondary to maternal toxicity and associated with growth retardation of foetuses. No treatment related effects were observed at 15 and 40 mg/kg b. wt/day dose groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related visceral anomalies were observed in foetuses of the treatment groups.
Other effects:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were observed in head razor sections from foetuses of the treatment groups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
LOAEL
Effect level:
110 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: skeletal growth retardation

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal growth retardation (high dose only)
Description (incidence and severity):
Considered secondary to overt maternal toxicity, morbidity and mortality that occurred in maternal animals (high dose only).

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
110 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In an OECD 414 prenatal developmental oral (gavage) toxicity study conducted according to GLPs, the maternal and developmental NOAELs for triphenyl phosphite were 40 mg/kg/day.

Executive summary:

In a guideline OECD 414 prenatal developmental toxicity study conducted according to GLP, triphenyl phosphite was administered by gavage on gestation days 5 -19 to groups of 25 female rats at dose levels of 0, 15, 40, and 110 mg/kg/day. Overt maternal toxicity, morbidity and mortality occurred at the high dose level of 110 mg/kg/day. Lower body weights and skeletal growth retardation were observed in fetuses at the high dose level, and are considered secondary to maternal toxicity. No treatment related effects were observed in maternal and developmental parameters at dose levels of 15 and 40 mg/kg/day. Based on these results, the maternal and developmental NOAELs for triphenyl phosphite are considered to be 40 mg/kg/day.

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