3,9-bis[2,4-bis(1-methyl-1-phenylethyl)phenoxy]-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane;bis(2,4-dicumylphenyl) neopentyl diphosphite

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-920-2

EC Name:

-

Cas Number:

154862-43-8

Molecular formula:

C53-H58-O6-P2

IUPAC Name:

3,9-bis[2,4-bis(2-phenylpropan-2-yl)phenoxy]-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

Specific details on test material used for the study:

TEST ITEM NAME: 3,9-bis[2,4-bis(1-methyl-1-phenylethyl)phenoxy]-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Dover Chemical Corporation, Lot No. 46D030816
- Expiration date of the lot/batch: March 08, 2017
- Purity: 99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In original container in a dry/cool, well ventilated place.
- Solubility and stability of the test substance in the solvent/vehicle: stable in vehicle for 4 hours.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 13 wks

- Housing: individually, except during the mating period. During the mating period, the rats were housed in groups of 2 rats/cage (one male plus one female). Confirmed mated female rats were caged individually, and nesting material was provided from 14th day of gestation. During the study, the rats were housed in solid floor polypropylene rat cages (size: 41 x 28.2 x 18 cm). Each cage was fitted with a stainless steel top grill having provision for keeping a polypropylene water bottle with stainless steel drinking nozzle. The bottom of the cages were layered with clean sterilized rice (paddy) husk as the bedding.
- Diet (e.g. ad libitum): standard rodent pellet feed (Teklad Certified Global 16% Protein Rodent diet manufactured by Envigo, USA) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24 deg C
- Humidity (%): 64 - 67%
- Air changes (per hr): 20-21
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test dose formulations were prepared every day prior to dosing and were used within 4 hours of preparation. The prepared formulation was kept on a magnetic stirrer in order to maintain a homogeneous preparation. The dose volume was 10 mL/kg body weight.

Details on mating procedure:

Each female was placed with a single male from the same dose level until copulation occurred or 2 weeks had elapsed. Each morning, the females were examined for the presence of sperm and the ‘day 0’ of pregnancy was recorded. Day 0 of pregnancy was defined as the day on which sperm was observed in the vaginal smears of rats.

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 dose formulation was analysed using a validated HPLC method once before initiation of treatment and twice during treatment period. Dose formulations were found to be within the acceptance level of ± 15% of nominal concentration demonstrating that the prepared concentrations were as intended by the study plan and %CV was less than 10, suggesting that the prepared formulations were homogeneously mixed.

Duration of treatment / exposure:

Dosing of both the sexes was initiated 2 weeks prior to mating and continued during the mating period. After mating, the male rats were further dosed up to and including the day before scheduled sacrifice (after approximately 80% of the females had delivered ). Females were further dosed during pregnancy and up to post-partum day 3.

Frequency of treatment:

once daily, seven days a week, at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

500 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:

15 males and 15 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of 14-day dose range finding study (JRF Study N° 410-1-04-14642), in which no test item related effect was observed in high dose (i.e., 1000 mg/kg b. wt./day) on body weight, feed consumption and organ weight.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice a day for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day for visible clinical signs during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of all the male animals were recorded on the first day of dosing and at weekly interval thereafter and at death. Body weight of all female animals was recorded on first day of dosing and at weekly interval during pre-mating and mating period. During gestation period, female animals were weighed on gestation days 0, 7, 14 and 20. During lactation period, female animals were weighed within 24 hours of parturition (day 0 post-partum), post-partum day 4 (i.e., lactation day 4) and at death.

FOOD CONSUMPTION:
- Feed consumption of all male animals was measured weekly during pre-mating and post-mating period. For female animals, during pre-mating period feed weights were recorded at weekly interval. During gestation period, feed weights were measured on days 0, 7, 14 and 20 and during lactation period, feed weights were measured on lactation days 0 and 4. Feed consumption was not measured during mating period.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number of pups, sex of pups, stillbirths, runts and the presence of gross anomalies. Pups which were found dead in the study were weighed and subjected to post-mortem examination.

Individual pup body weight was recorded on lactation days 0 and 4.

Postmortem examinations (parental animals):

All animals were subjected to full gross necropsy. At the time of sacrifice, the testes and epididymis of all adult male animals were weighed. During sacrifice, number of corpora lutea and implantation sites were recorded for each dam.

Detailed histological examination was performed on the ovaries, testes and epididymis of the animals from the high dose group and the control group.

Postmortem examinations (offspring):

All pups were subjected to gross pathological examination.

Statistics:

Data such as body weight, body weight change, feed consumption, uterine data (number of implantation, number of corpora lutea, the mean number and percent of pre-implantation, post-implantation and post-natal loss), litter parameters (number and weight of male pups, female pups, total pups (male + female)), duration of gestation and pre-coital interval were analysed using Bartlett’s test of homogeneity of variance. If the result was not significant then analysis of variance (ANOVA) was carried out and if the results was significant then t-test was carried out. If ANOVA was significant then Dunnett’s multiple comparison tests was carried out.
Data such as mortality rate, gestation index, parturition index, pups survival index, live birth index and fertility index were analyzed using Chi-Square test.

Reproductive indices:

male fertility index, female fertility index, gestation index, parturition index and mating index, gestation period and pre-coital interval, pre-implantation loss, pre-natal (post-implantation) loss, post-natal loss.

Offspring viability indices:

live pups index and live birth index, survival / mortality index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no effects observed at limit dose of 1000 mg/kg/day

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 other: maternal dose (mg/kg/day, actual dose received)

Sex:

male/female

Basis for effect level:

other: no effects observed at highest dose tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Parental animals

There were non-treatment-related mortalities. There were no effects on the following parameters in parental animals: clinical signs, body weight, food consumption, organ weights, gross pathological effects, histopathological effects, reproductive performance, duration of pregnancy, pre-implantation loss, parturition index, or live birth index.

F1 animals

There were non-treatment-related mortalities. There were no effects on the following parameters in F1 animals: clinical signs, food consumption, fetal body weight changes, reduction in number of live offspring, changes in sex ratio, changes in litter size and weights, changes in postnatal survival and external malformations.

Applicant's summary and conclusion

Conclusions:

Daily oral gavage administration of the 3,9-bis[2,4-bis(1-methyl-1-phenylethyl)phenoxy]-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane(CAS: 154862-43-8) to male and female rats at dose levels of 250, 500 and 1000 mg/kg b. wt./day during pre-mating, mating, post-mating, gestation period and until post-partum day 3 did not produce test item-related adverse effects on reproduction and development. Thus, the No Observed Adverse Effect Level of the substance CAS: 154862-43-8 was found to be 1000 mg/kg b. wt./day.