3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

EC Number:

243-869-3

EC Name:

3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide

Cas Number:

20544-37-0

Molecular formula:

C19H22O6P2

IUPAC Name:

3,9-dibenzyl-2,4,8,10-tetraoxa-3λ⁵,9λ⁵-diphosphaspiro[5.5]undecane-3,9-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

CAS No.: 20544-37-0
Lot number: 44216018
Appearance: Colourless Powder
Purity: 99.9%
Expiry Date: October 25, 2023
Storage conditions: Room temperature (20-30°C)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were procured from CPCSEA approved vendor [Hylasco Biotechnology (India) Pvt. Ltd., Telangana]
- Females nulliparous and non-pregnant: yes
- Age at study initiation: At least 13 weeks at the start of estrous cycle evaluation
- Weight at study initiation: (P) Males: 413 - 547 g; Females: 226 - 302 g; (F1) ~ 5 - 9 g
- Fasting period before study: none
- Housing: Two to three rats were housed in each polycarbonate cage (length 37 cm X breadth 21 cm X height 20 cm). Pregnant females were housed individually.
- Diet: conventional laboratory pellet diet (Batch no. 040519, 040719 and 040919) from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum.
- Water: Aquaguard™ filtered drinking water was offered ad libitum in bottles.
- Acclimation period: Animals were acclimatised to the test conditions for at least 5 days prior to estrous cycle evaluation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.10 and 23.00
- Humidity (%): 40.20 and 65.70
- Air changes (per hr): 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 (w/v)Methylcellulose

Details on exposure:

The groups received 0, 100, 300 and 1000 mg/kg body weight of test-item respectively.
Dose administration was through oral gavage.
Tthe vehicle used in this study was 0.5% solution of Methyl cellulose.
Dosing of both sexes were started 2 weeks prior to mating, after they had screened for normal estrous cycle (in a 2 weeks pre-treatment period). Dosing was continued in both sexes during the mating period.
All males were administered with dose formulation for a total of 28 days before their scheduled sacrifice.
Females were further exposed to test item during gestation and up to, and including, day 13 post partum or a day before terminal sacrifice.
Non parturated females were sacrificed on gestation day 36.
To ensure accurate administration of test-item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses on two occasions – Day 6 and Day 31 of treatment.

Details on mating procedure:

During mating period, one male and one female animal from the same dose group were cohabited. Mating was confirmed by presence of sperm in vaginal smear. Females showing sperm positive smear were separated and housed individually. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

females: 55 days
males: 28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All animals were observed daily for clinical signs and symptoms after dose administration. These observations were regularly performed approximately 1 hour after completion of dose administration, since onset of clinical signs after oral gavage is anticipated in that time frame.
All animals were subject to a detailed clinical examination once before the first treatment (to allow for within-subject comparisons) and weekly thereafter throughout the treatment period. The animals were examined outside the home cages and the observations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions & excretions and autonomic activity (eg. lacrimation, piloerection, pupil size, respiratory pattern). The gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also checked for and recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once on receipt, once during randomization, once at the start of treatment and once every week thereafter, till the end of the experimental period. During pregnancy, females were weighed on gestation days 0, 7, 14, 20 and on the day of parturition (lactation day 0). Further, females were weighed on lactation day 4, 7 and 13.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, The weight of feed provided and that leftover was recorded for each cage once every week starting from Day 1 of the treatment phase. This data was used to calculate mean feed consumption by each animal over weekly periods. Additionally, feed consumption for females were recorded on gestation day 7, 14 and 20 and on lactation day 4, 7 and 13.
BODY WEIGHT: Yes, animals were weighed once on receipt, once during randomization, once at the start of treatment and once every week thereafter, till the end of the experimental period. During pregnancy, females were weighed on gestation days 0, 7, 14, 20 and on the day of parturition (lactation day 0). Further, females were weighed on lactation day 4, 7 and 13. Data of body weight was used to compare absolute weights as well as the percent change in weights of the animals through the course of study . Animals fasted overnight prior to euthanasia and were weighed before necropsy. T and this data served as a calculation basis for normalisation ofrelative organ weights for each animal.
CLINICAL BIOCHEMISTRY:
Animals were overnight fasted prior to blood collection. Blood from two sacrificed pups (one female and one male ) per litter on day 4 after birth (if the number of pups allowed), from all dams and at least two pups per litter (one female and one male) at termination on day 13 and from all adult males at termination were collected and stored under appropriate conditions. From all parent males, females and PND 13 pups blood was collected from retro orbital plexuses whereas from PND 4 pups blood was collected through cardiac puncture and pooled by litter. All blood samples collected in the study were assessed for serum levels of thyroid hormones (T4) using Rat Thyroxine (T4) ELISA kit (KinesisDx USA).,

Oestrous cyclicity (parental animals):

Estrous cycle was monitored daily for two weeks prior to treatment initiation. It was also assessed daily from the beginning of the treatment and continued until confirmation of mating. Vaginal smears were observed in the morning of scheduled sacrifice to determine the stage of estrous cycle for better correlation with histopathology of ovaries. Vaginal smear was taken carefully to avoid disturbance of vaginal mucosa.

Litter observations:

Each litter was examined after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities.
Pups found dead on PND 0 or at a later time were examined for possible defects and cause of death.
Live pups were counted and weighed individually as soon as possible on PND 0, and regularly thereafter, including PND 4, 7 and 13. Clinical examinations of pups were carried out on PND 0, 4, 7 and 13. Signs noted included but limited to external abnormalities, changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour, were also checked for and recorded.
The anogenital distance (AGD) of each live pup was measured on PND 0. The AGD of each pup was normalised by its size (cube root of body weight). The presence of nipples/areolae in male pups were checked and counted on 13.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: At termination or death during the study, all the animals including pups were examined macroscopically for any pathological changes and anomalies. Time of death of each animal is recorded. Special attention was paid to the organs of the reproductive system. All adult animals at termination were euthanized by CO2 asphyxiation. Pups at termination were euthanized by cervical dislocation following CO2 narcosis. Dead pups and pups sacrificed on day 13 post-partum were carefully examined externally for gross abnormalities, especially regarding external reproductive genitals.
The number of implantation sites was recorded from all females.
ORGAN WEIGHT: Organs were weighed immediately after they were collected. Organs were kept in normal saline till they were weighed (after having carefully removed/ dried the saline). In case of paired organs, combined weight was recorded. From two pups per litter (one pup/ sex/ litter where possible) thyroid gland was collected at termination on day 13 and weighed after fixation.
HISTOPATHOLOGY: The following tissues of all adult animals of all groups G1-G4 were preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed using Modified Davidson fluid) for subsequent histopathological examination:
Adrenals, Prostate gland, Epididymides, Seminal vesicle with coagulating glands as a whole, Ovaries with oviduct, Testes, Levator ani plus bulbocavernosus muscle complex, Thyroid gland with parathyroids, Cowper’s glands, Uterus with cervix, Glans penis, Vagina, Pituitary gland.

Postmortem examinations (offspring):

Full histopathology was carried out on the preserved organs and tissues of all animals in the control and high-dose groups of both sexes. Additionally, Tthe thyroid glands from pups and from the adults animals were histopathologically examined in control and high dose groups .

Statistics:

Raw data were analysed using the statistical software “Sigma Plot 14.0” (supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data were summarized in tabular form. All continuous data (body weight, feed consumption, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality statistically relevant differences using Shapiro Wilk test. All homogenous data were analysed using ANOVA and data showing statistical significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data were analysed using Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs or symptoms were observed in any of the animals of either sex through the study period.
All adult animals seemed physiologically normal with respect to all the parameters examined during detailed clinical examinations through the course of the study. No treatment related symptoms were found up to the dose level of 1000 mg/kg body weight in either sex.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22 and 29) recorded during the 28 days exposure period for males. No significant difference was found for the mean body weights of female during two weeks pre-mating period, during mating, during gestation and during lactation period.
The percent change in body weights with respect to the first day of exposure was also statistically comparable among all the groups of males and females. Moreover, the percent changes in body weights with respect to gestation day 0 and lactation day 0 were also calculated and the results were statistically comparable across the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The feed consumed per animal per day remained statistically comparable among the experimental groups within each sex at every point of observation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone (T4) levels in sera were found to be statistically comparable among the control and treatment groups in adult males, PND 4 pups and in PND 13 male and female pups. Thyroid levels of the adult females were however found to be increased in G3 (P<0.05) as compared to G1 animals, while it remained comparable in other dose groups. This observation was inconsistent with the dose levels and therefore not of great informative value, in the context of the current study.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related histopathological findings are reported in this study that could arise out of test-item administration. All observed tissues were normal at the level of histology. Minimal and focal lesions were observed in some slides of testes, adrenal, and uterus, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity.
Keeping in line with the study plan, since the results of histopathology evaluation did not point to any treatment-related effects in any of the organs, the microscopic examination of the low- and mid-dose groups was not carried out.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle was monitored in females for its regularity. All females received for the study displayed a regular estrous cyclicity of 4-6 days, before they were randomly allocated to the experimental groups. During two weeks of pre-mating treatment period, estrous cycle length remained unaltered among the groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating and pregnancy behaviour:
Females of all groups showed presence of sperm in vaginal smear during cohabitation period. Precoital interval (pairing to insemination) was less than 5 days for all the females, except one G3 and one G4 female which showed sperm positive smears after 6 days of cohabitation. However, the precoital interval was statistically comparable amongst all groups.
There was no statistical difference between the control (G1) and treatment groups (G2, G3 and G4) with respect to gestational length, litter size, number of live births and post-implantation loss. Although the presence of sperm was evident in all the females, no implantation sites were observed in 2 females from G1, 1 from G2, 2 from G3 and 1 from G4 at the time of terminal sacrifice. These females have been indicated as non-parturated females in the given data.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

reproductive function (oestrous cycle)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related anomalies were observed in pups at the time of gross observation on post-natal day (PND) 0. Sex ratio of pups (Male to Female) remained comparable at PND 0 and PND 4 for all dose groups.
No treatment-related clinical signs or symptoms were observed in any of pups of either sex during the lactation period.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When individual pup body weights were analysed, a significantly elevated group mean was observed for G4 (p<0.05) in comparison with the control group on PND 0.
Similarly, mean pup body weight on PND 4 was higher in G3 (P<0.001) and G4 (P<0.01) groups as compared to control group.
On PND 7, notably increased body weight was observed only in G3 (P<0.001) group against the control group.
Mean pup body weight on PND 13 was significantly higher in G3 (P<0.001) and G4 (P<0.001) group a s compared to G1 animals.
Although, pup body weight was significantly elevated at all the stated time points, the differences did not follow a dose-dependent pattern.
Moreover, when the same data was analysed by keeping litter as a unit of comparison, the body weights at all the instances were equivalent among the groups, thus indicating that the significant increase observed in individual pup body weights was very likely merely due to a large sample size and had little or no relevance biologically.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone (T4) levels in sera were found to be statistically comparable among the control and treatment groups in adult males, PND 4 pups and in PND 13 male and female pups.
Thyroid levels of the adult females were however found to be increased in G3 (P<0.05) as compared to G1 animals, while it remained comparable in other dose groups. This observation was inconsistent with the dose levels and therefore not of great informative value, in the context of the current study.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (AGD) was measured in all the pups on their respective PND 0.
AGD was significantly higher in male pups of G4 (P<0.01) as compared to the respective controls. A dditionally, the AGD in pups was normalised to its weight (cube root of pup body weight) and analys ed for statistical differences. Normalised AGD was also found to be increased in male pups of G4 (P <0.05) as against the G1 males.
In case of females pups, AGD was reduced in G2 (P<0.05) as compared to control group, whereas it remained comparable to control (G1) in G3 group. When analysed for normalised AGD in female pups, significant decrease was found in all the treatment groups G2 (P<0.001), G3 (P<0.01) and G4 (P<0.01) as compared to control group. However, as observed in case of pup body weight, when litter was used as a unit for comparing AGD data, the means were comparable for all groups and with the available literature.

Description (incidence and severity):

Nipples/areolae were absent in all male pups at PND 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Upon gross necropsy examination, no abnormality was observed in the found dead pups and in the terminally sacrificed animals including males, females, PND 4 pups and PND 13 pups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from this study and under the experimental conditions used in this study, it is concluded that the Reproduction/ Developmental No Observed Adverse Effect Level (NOAEL) of the test item 3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide found to be 1000 mg/kg body weight, when administered daily by oral gavage to Sprague Dawley rats over a period of 28 days for males and up to 56 days for females.

Executive summary:

The results of this study reveal that 3,9-dibenzyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide does not cause mortality up to a dose of 1000 mg/kg body weight when administered orally over a period of 28 days for male and up to 56 days for females. Further, no clinical signs or symptoms were observed in any animals of the study. Body weight, body weight change and feed consumption remained comparable among the groups for every time point recorded through the study period and with the available reference data.

All the treated animals demonstrated regular estrous cycle of 4-6 days and most of the animals were mated within 5 days of cohabitation. Further, gestation length, litter size, live births, post-natal loss, survival index and sex ratio remained comparable among all the groups.

No anomalies were observed in pups at the time of gross observation on PND (post natal day) 0. Sex ratio of pups remained comparable at PND 0 and PND 4 for all dose groups. Body weight of pups at PND 0, 4, 7 and 13 was statistically higher in the high dose group (G4) as compared to control group (G1). However, litter weight at all these occasions was comparable amongst the groups.

Mean normalised AGD (anogenital distance) was found to be increased in male pups of high dose group G4, whereas no difference was observed in other groups compared to the control group. Mean normalised AGD of female pups was significantly lower in all treatment groups (G2, G3, and G4) as compared to control group (G1).

Although, pups body weight and AGD differ statistically between G1 and treatment groups, this is likely an outcome of a high number of samples therein. It is not uncommon to find biologically comparable values differing statistically due to high number of samples. Also, the AGD and mean body weight at all instances were well within their respective biological range when compared with the available literature.

Moreover, since no statistically significant difference was found in the group means of both these parameters when litter was used as the unit of comparison, and since these findings are not supported by any other histopathological observations which would support a test item related concern, these findings cannot be attributed to test item application.

T4 levels remained comparable among all groups of parent males, PND 4 pups, PND 13 male and female pups. Parent females showed increased levels of T4 in G3 animals as compared to control animals of G1. This increase is deemed unrelated to test item administration since the mean of G4 was statistically comparable to that ofcontrol.

Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test item administration over all treatment groups.

No effect of test item was observed on absolute or relative organ weights of parent males and PND 13 females. The mean weight of ovaries was reduced in parent animals of G4 group as compared to control, whereas thyroid and parathyroid weight was increased in G4 group of PND 13 male as compared to control. However, these changes in organ weights in G4 animals do not correlate with any histological observations made, making it inconsequential from the point of view of toxic effects of test item.

Histopathological observations were made for G1 (vehicle control) and G4 (high dose) animals and apart from very few sporadic findings in both groups, no treatment related changes showed up in any of the observed tissues at the tested dose (1000 mg/kg body weight) in either sex.

Taking into consideration overall findings herein, it is not possible to pin down any test item related or dose-dependent toxic effect under this study design.