Phosphorous ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2009-01-30 to 2009-08-14

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

esterification products of castor oil and polyphosphoric acids

EC Number:

614-406-6

Cas Number:

68308-61-2

Molecular formula:

C57H107P3O18

IUPAC Name:

esterification products of castor oil and polyphosphoric acids

Details on test material:

- Name of test material (as cited in study report): Gardo TP10451
- Physical state: Liquid (brown, inconspicuous odour)
- Analytical purity: 100% (titration)
- Purity test date: 2008-03-20
- Lot/batch No.: 2939R
- Expiration date of the lot/batch: 2010-02-18
- Stability under test conditions: Stable at room temperature, will decompose when heated above 90 °C
- Storage condition of test material: Stored in a dry, cool place

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd., 1103 Budapest, Cserkesz u. 90
- Age at study initiation: (P) 10 wks;
- Weight at study initiation: (P) Males: 343-466 g; Females: 212-270 g
- Fasting period before study: no fasting period
- Housing: Before mating: 4 animals of the same sex / cage; Mating: 1 male and 1 female / cage
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water: tap water from municipal supply, as for human consumption from 500 mL bottle, ad libitum
- Acclimation period: 35 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 24.8 °C
- Humidity (%): 24 - 50 %
- Air changes (per hr): 15-20 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours light daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

The test item was formulated in PEG400 in concentrations of 12.5, 62.5 and 250 mg/mL. Formulations were prepared in the Central Dispensary of LAB Research Ltd. for use within 96 hours, and were stored refrigerated pending use. Assessment of test item stability in this vehicle, in the conditions employed on the study indicated an up to 6 hr stability at room temperature, and 96 hr stability at 2-8°C, at concentrations from approximately 1 to 250 mg/mL in PEG400 formulations, with a recovery within the acceptable range of 100 ± 10% (104-94%).
Dose formulation samples were collected and analysed for concentrations achieved and homogeneity by the Analytical Laboratory of LAB Research Ltd. during the first and last weeks of treatment (on 19 March and 28 April 2009). Dose formulations were homogenous. No GARDO TP10451 was detected in the control samples; in the test item dose formulations, measured concentrations ranged from 102 to 109 % of nominal concentrations. As this test item is not soluble in water, PEG400 was considered the suitable vehicle to use for the preparation of dose formulations for oral administration.
A constant treatment volume of 4 mL dose formulation/kg bw was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals determined at least weekly. In the first week of the pre-mating period, animals received the volumes based on the actual body weight on day 0.

Details on mating procedure:

Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred (up to 8 days). Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope, the presence of the vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421, or gestation day GD0). Sperm positive females were caged individually. Mating pairs were clearly identified in the data and are presented in the study report; mating of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Assessment of test item stability in this vehicle, in the conditions employed on the study indicated an up to 6 hr stability at room temperature, and 96 hr stability at 2-8°C, at concentrations from approximately 1 to 250 mg/mL in PEG400 formulations, with a recovery within the acceptable range of 100 ± 10% (104-94%).

Duration of treatment / exposure:

Dosing of both sexes began after an appropriate acclimatisation period and two weeks before mating and was continued up to and including the day before necropsy.
Males were dosed for 28 days, 14 days pre-mating and 14 days mating/post- mating period , then they were euthanised and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 8 days mating period, through gestation (up to 23 days) and day 3 post-partum with necropsy the following day (up to treatment day 47). The day of birth (viz. when parturition was complete) was defined as day 0 post-partum.

Frequency of treatment:

The test item was administered daily by oral gavage, at similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.

Details on study schedule:

see durantion of treatment / exposure.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 250 and 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Adult animals were identified under the study by tattoo numbers on the tail, which were cross referenced with LAB Research Ltd.'s master file number for each animal allocated to the study, as indicated in the Experimental Design section. The boxes were marked by identity cards, with information about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Boxes were arranged in such a way that possible effects due to cage placement were minimized. Marking of new-borns was performed by digit clipping for identification.
All parental (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that animals of all test groups were as nearly as practicable of uniform weight. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages.

Positive control:

no positive control.

Examinations

Parental animals: Observations and examinations:

Clinical Examination:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. All signs of toxicity were recorded including onset, degree and duration of signs. No obvious behavioural changes or signs of difficult or prolonged parturition were noted. Weekly, more detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy.

Weight Assessment:
All parent animals were weighed with accuracy of 1 g on the first day of dosing (day 0), at least weekly thereafter and at termination. Parent females were weighed on gestation days (GD) 0, 7, 14 and 20 and on post partal days PND 0 (within 24 hours after parturition) and 4 before termination. Body weight of the female animals was additionally measured on GD 10 and 17 in order to give accurate treatment volumes, but these data are not evaluated statistically. Body weight data are reported individually for adult animals.

Food Consumption:
The food consumption was determined at least weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment periods.

Observation of the Delivery Process, Offspring and Nursing Instinct:
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded. There was no evidence of abnormal deliveries. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed for nesting behaviour (whether they made a nest from the bedding material and cover their new-borns or not). The efficiency of the suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that were significantly smaller than normal pups; no such pups were observed), and the presence of gross abnormalities, if any.
Live pups were counted, sexed, weighed individually with an accuracy of 0.1 g within 24 hours of parturition (on the first day after parturition was complete), and on day 4 post partum. Observations are reported individually for each adult animal.
In addition to the observations on parent animals, behaviour of the offspring was evaluated. No abnormal behaviour was noted. All the litters were checked and recorded daily for the number of viable and dead pups. The pups that were found dead were cannibalised, thus, they were counted and sex determined if possible, but not further examined macroscopically.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Special emphasis was made on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

- Mean pup body weight (per pup and per litter) on postnatal days 0 and 4
- Mean pup body weight gain (per litter) between postnatal days 0 - 4
- Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4

Postmortem examinations (parental animals):

Pathology:
Gross necropsy was performed on each animal irrespective of the date of its death. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia by exsanguination. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Any abnormality was recorded with details of the location, colour, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The testes, epididymides (total and cauda), seminal vesicles and prostate, and the female reproductive organs including ovary, uterus (with and without cervix), vagina, and brain and pituitary of all adult animals were weighed. Paired organs were weighed separately.
The weighed organs and all organs showing macroscopic lesions, of all adult animals were preserved. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10 % buffered formalin solution.
Pups euthanized at day 4 postpartum were carefully examined at least externally for gross abnormalities. No pups with abnormalities in structure or behaviour were observed.

Histopathology:
Detailed histological examination was performed on the ovaries, uterus, vagina, pituitary, testes and epididymides of the animals in the control and high dose groups, and all gross lesions.
Special emphasis was made on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Due to apparently higher incidence of epithelial apoptosis noted in the high dose group, additional histology evaluation was performed on the testes and epididymides of all the male animals (low and mid dose).

Postmortem examinations (offspring):

Pups euthanized at day 4 postpartum were carefully examined at least externally for gross abnormalities.

Statistics:

Statistical Evaluation:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to assess the significance of inter-group differences. Where significant result was obtained at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as required.

Reproductive indices:

- Male mating index
- Male fertility index
- Female mating index
- Female fertility index
- Gestation index
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)

Offspring viability indices:

- Survival index of pups on postnatal days 0 and 4
- Sex ratio % (on postnatal days 0 and 4).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

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

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

GARDO TP10451 administered daily by oral gavage in Wistar rats, for 28 days in the male animals, and up to 47 days (females), at dose levels of 50, 250 and 1000 mg/kg bw/day, did not result in any clinical changes that could be ascribed to the treatment.
No effects considered adverse, or toxicologically significant, were noted on the mean body weight, body weight gain, or food consumption values in the treated groups compared to control animals. Treatment related effects were observed in the body weight gain of females in the high dose group. These effects were nevertheless not regarded as adverse, taking into account the absence of other treatment related effects at this dose level.
There were no significant differences between the control and test item treated groups with regard to reproductive ability, or in the mating, fertility or gestation indices. Test item administration did not impact the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 8 days of pairing (cohabitation). No test item effect on the duration of pregnancy was observed. All females littered in 22 to 23 days. There were no abnormalities in pups that were ascribed to the treatment. All the parturitions were normal. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or post-partal period noted in the treated groups at dose levels up to and including 1000 mg/kg bw/day compared to control animals. There were no test item-related alterations in the delivery data of GARDO TP10451 treated dams as compared to the control value. The number of corpora lutea and implantations were comparable or slightly higher in the treated groups up to and including 1000 mg/kg bw/day, compared to females in the control groups. Pre-implantation, intrauterine and total mortality values were lower in the high dose group administered 1000 mg/kg bw/day than in the control animals. Slightly higher values than control without attaining statistical significance were observed at 50 and 250 mg/kg bw/day; however, the differences were minor, and based on the lack of similar changes in the high dose group, these variations were neither considered toxicologically significant, nor related to test item administration. In the parent animals surviving to scheduled termination, the administration of GARDO TP10451 at dose levels of 50, 250 or 1000 mg/kg bw/day was not associated with any adverse test item-related macroscopic or microscopic findings. There were no treatment-related organ weight changes. Treatment related effects were observed in apoptosis parameters in the epididymides in males in the high dose group. These effects were nevertheless not regarded as adverse in the absence of an impairment of any reproductive performance parameters in the high dose group.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: based on female body weight reductions and apoptosis in the epididymides of males in the high dose group

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: based on the absence of toxicologically adverse effects up to and including the high dose level of both sexes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Litter examination did not reveal any clinical, or macroscopic test item-related effects compared to observations noted in the control group. GARDO TP10451 administration had no adverse effect on the mean or total number of pups, or pup survival index. The sex ratio was similar in the control and treated groups, and no significant variations were observed on PN4 vs. PN0.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no adverse effects observed on litter

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study, a no observed effect level (NOEL) for parental effects (systemic) of 250 mg/kg bw/day was derived for GARDO TP10451 based on female body weight reductions and apoptosis in the epididymides of males in the high dose group. The no observed adverse effect level (NOAEL) for GARDO TP10451 for parental effects (systemic) was 1000 mg/kg bw/day based on the absence of toxicologically adverse effects up to and including the high dose level of both sexes. For reproduction parameters, no effects were noted at any dose level, resulting in a NOAEL of 1000 mg/kg bw/day. For pup growth rate and adverse effects, the NOAEL was 1000 mg/kg bw/day.

Executive summary:

GARDO TP10451 administered daily by oral gavage in Wistar rats, for 28 days in the male animals, and up to 47 days (females), at dose levels of 50, 250 and 1000 mg/kg bw/day, did not result in any clinical changes that could be ascribed to the treatment. No effects considered adverse, or toxicologically significant, were noted on the mean body weight, body weight gain, or food consumption values in the treated groups compared to control animals. The parental animals displayed no effects related to treatment with regard to the reproductive ability and mating, gestation, parturition or post-partal period. There were no adverse findings at macroscopic or microscopic examination at up to and including 1000 mg/kg bw/day. There were no treatment-related organ weight changes.

Treatment related effects were observed in the body weigh gain of females and in apoptosis parameters in the epididymides in males both in the high dose group. These effects were nevertheless not regarded as adverse, taking into account the absence of other treatment related effects at this dose level for both sexes.

In the F1 generation, there were no pups with adverse clinical changes that could be ascribed to test item administration, or gross abnormalities. No toxicologically significant effect on pup body weight, or pup survival was noted.

In conclusion, under the conditions of this study, a no observed effect level (NOEL) for parental effects (systemic) of 250 mg/kg bw/day was derived for GARDO TP10451 based on female body weight reductions and apoptosis in the epididymides of males in the high dose group. The no observed adverse effect level (NOAEL) for GARDO TP10451 for parental effects (systemic) was 1000 mg/kg bw/day based on the absence of toxicologically adverse effects up to and including the high dose level of both sexes. For reproduction parameters, no effects were noted at any dose level, resulting in a NOAEL of 1000 mg/kg bw/day. For pup growth rate and adverse effects, the NOAEL was 1000 mg/kg bw/day.