tetrakis(2,6-dimethylphenyl)-m-phenylene biphosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2011 - 11 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to current OECD guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Wistar rat was selected due to experience with this strain of rat in reproduction toxicity studies and known fertility.Young adult rats, approximately 10 weeks old at starting and 12 weeks at mating. The age range within the study was kept to the minimum practicable.Rodents were group-housed as practical, up to 5 animals/ group/cage (paired or single-housed during the mating and gestation/delivery period, respectively). Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities (i.e. nesting).

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

The dosing solutions were administered daily by oral gavage, using a tipped gavage needle attached to a syringe. A volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. The test item was administered daily by oral gavage on a 7 days/week basis. Control animals were treated concurrently with the vehicle only. Dosing of both sexes began after a minimum of five days acclimatisation (A) and 2 weeks before mating and continued up to and including the day before the necropsy. Mating began after the animals have attained full sexual maturity and continued in both sexes during the mating period.

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 4 days. A vaginal smear was prepared daily for each female during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last week and midway (29 November 2011, 21 December 2011 and 09 January 2012), one set to analyse, collected in replicates as practical, and one set as a back-up, which was not required for confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.

Duration of treatment / exposure:

1/Control002/Low dose5012.53/Mid dose25062.54/High dose1000250

Frequency of treatment:

Females were dosed for 14 days pre-mating, for up to 4 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum dosing. The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. There was no female during the study that showed no-evidence of copulation (non-mated). Not-delivered female 2502 was sacrificed 26 days after the last day of mating, as practical. Female 4503 was found dead and underwent necropsy with macroscopic examination on Day 11, before the onset of mating.

Details on study schedule:

Details of schedule attached to technical dossier.The dosing solutions were administered daily by oral gavage, using a tipped gavage needle attached to a syringe. A volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. The test item was administered daily by oral gavage on a 7 days/week basis. Control animals were treated concurrently with the vehicle only. Dosing of both sexes began after a minimum of five days acclimatisation (A) and 2 weeks before mating and continued up to and including the day before the necropsy. Mating began after the animals have attained full sexual maturity and continued in both sexes during the mating period.

Remarks:

Doses / Concentrations:50Basis:nominal conc.peanut oil

Remarks:

Doses / Concentrations:250Basis:nominal conc.peanut oil

Remarks:

Doses / Concentrations:1000Basis:nominal conc.peanut oil

No. of animals per sex per dose:

48 male, 48 female rats and sufficient spare animals/sex, 12 male and 12 female rats/group, 4 groups, were assigned to the study

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Females were allowed to litter and rear their offspring. Delivery process was carefully observed. All observations were recorded and the animals monitored for any evidence of abnormal deliveries. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of 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 are significantly smaller than normal pups) and the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (on the first day after parturition was complete, i.e. Day 0 or 1 post-partum) and on Day 4 post partum with an accuracy of 0.01g.Observations are reported individually for each adult animal. In addition to the observations on parent animals, the offspring F1 generation was examined daily for the number of viable and dead pups or any clinical or behavioural abnormalities.

Oestrous cyclicity (parental animals):

A vaginal smear was prepared daily for each female during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Litter observations:

All F1 offspring were terminated on Day 4 post-partum.

Postmortem examinations (parental animals):

Gross necropsy was performed on each animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia (details are presented in "Details of Other Materials") by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.At the time of termination, body weight and weight of the following organs of all parental animals were determined:- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain- With a precision of 0.001 g: ovaries, pituitary Paired organs were weighed individually; absolute organ weights were measured and are reported. Relative organ weights (to body and brain weight) were calculated and are reported. The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution.

Postmortem examinations (offspring):

Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities. Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups, found dead female 4503, and any macroscopic findings (abnormalities) observed. Special attention was paid to evaluation of the 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.The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. No additional examination was required, as no test item related findings were noted.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

One Group 4 female, 4503 was found dead on Day 11, before the onset of mating, due a dosing accident, unrelated to the test item. As of Day 8, prior to its death, clinical signs including red liquid from the nostrils and hunched back position were noted, followed by decreases in activity on Day 10, then death on Day 11. At necropsy, oesophagus perforation confirmed the dosing accident, associated with white, creamy material in the thoracic cavity, dark red discoloration noted in the lungs and thickened rough surface in the pericardium and pleura.During the pre-mating period, the body weight of the female animals was 5.1% lower than control at 250 mg/kg bw/day, p<0.01 and 6% lower, at 1000 mg/kg bw/day, p<0.01, with statistically lower body weight gains during the second week of treatment between Days 7 and 14 as well as when evaluated for the whole pre-mating period between Days 0 and 14 (-64% and -67% lower, p<0.01, at 250 and 1000 mg/kg bw/day, respectively). Statistically significant, slightly lower than control mean body weight values were also recorded during the gestation period, 4.7% and 5% lower, p<0.01 on GD0, and 3.8% and 5.9%, p<0.05 and p<0.01 on GD4, at 250 and 1000 mg/kg bw/day, respectively. The body weight gain values of the pregnant females during the gestation period were however comparable with, or slightly higher than control, without attaining statistical significance. During the post-partum period, 5.3% lower than control mean body weight was noted on PPD4 at 1000 mg/kg bw/day, p<0.03, and only 2.7% lower than control at 250 mg/kg bw/day, without attaining statistical significance at this dose level. The body weight gains remained however lower than control at 250 and 1000 mg/kg bw/day during the post-partal period evaluated, between PPD0 and PPD4, without attaining statistical significance, with mean values of 6.93 and 1.36 g in the Mid and High dose groups, respectively, vs. 11 g in the control.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

There were no adverse effects on pups viability or any clinical observations recorded in the F1 generation that could be considered toxicologically relevant, or related to test item administration. The mean and total number of viable pups as well as pups survival indices showed no statistically or toxicologically significant differences to control. There were no pups found dead and intact. A low number of pups were found cannibalized, 1/153, 1/151, 2/154 and 2/131 in the control, low, mid and high dose groups, respectively, and 1/154 mid dose pups was not suckled on PND4; these minor variations were regarded as incidental and ascribed to biological variability, unrelated to PX-200 administration. There were no abnormalities at the external examination of all pups surviving to PND4. The sex ratios (%) were similar in the control and treated groups, with slightly lower values in the high dose group, however, without attaining statistical significance, and no variations were observed on PND4 compared to PND0. In the absence of statistically significant differences, the slightly lower sex ratio at 1000 mg/kg bw/day was considered equivocal in correlation to treatment and was regarded as reflecting biological variability and not a test item adverse effect under the conditions of this study.When compared to controls, the mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, were considered to show no toxicologically significant, adverse or test item related effects.Although statistically significant differences to control were noted, these were minor, comparable with the physiological values and showed no dose response or a consistent trend and were therefore considered incidental, biological variations.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Key result

Reproductive effects observed:

not specified

Summary of report

	Dose (mg/kg bw/day)
	Control	50	250	1000
Pairs started (N)	12	12	12	11$
Surviving females showing evidence of copulation (N)	12/12	12/12	12/12	11/11
Females achieving pregnancy (N)	12/12	11/12	12/12	11/11
Conceiving days 1 - 5 (N)	12	11	12	11
Pregnancy ≤ 21 days (N)	1	4	4	2
Pregnancy = 22 days (N)	11	7	8	9
Pregnancy ≥ 23 days (N)	0	0	0	0
Dams with live young born (N)	12	11	12	11
Dams with live young at PN4 (N)	12	11	12	11
Corpora lutea/dam (mean)	13.25	14.27	13.42	12.55
Implantations/dam (mean)	13.00	14.00	12.92	12.27
Live pups/dam at birth (mean)	12.75	13.73	12.83	11.91
Live pups/dam at day 4 (mean)	12.67	13.64	12.67	11.73
Sex ratio at birth (mean)	50.56	54.86	57.34	39.19
Sex ratio at PN4 (mean)	50.82	54.51	56.83	39.70
Litter weight (g) at birth (mean)	85.2	87.3	81.2	78.1
Litter weight (g) at day 4 (mean)	132.2	140.6	128.7	124.3
Pup weight (g) at birth (litter mean)	6.708	6.367	6.429	6.579
Pup weight (g) at day 4 (litter mean)	10.493	10.364	10.325	10.728
STRUCTURALLY ABNORMAL PUPS
Dams with 0/Dams with live born	12/12	11/11	12/12	11/11
Dams with 1 or ≥2	0	0	0	0
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	10/12	8/12#	9/12	9/11
Females with 1	1/12	3/12#	2/12	1/11
Females with 2	1/12	0/12#	0/12	1/11
Females with ≥3	0/12	1/12#	1/12	0/11
Pre-natal/post-implantations (implantation's minus live births) (intrauterine)
Females with 0	9/12	8/11	11/12	9/11
Females with 1	3/12	3/11	1/12	1/11
Females with 2	0/12	0/11	0/12	0/11
Females with ≥3	0/12	0/11	0/12	1/11
Post-natal (live births minus alive at post-natal day 4)
Females with 0	11/12	10/11	11/12	10/11
Females with 1	1/12	1/11	0/12	0/11
Females with 2	0/12	0/11	1/12	1/11
Females with ≥3	0/12	0/11	0/12	0/11

Conclusions:

The no observed effect level NOEL and no observed adverse effect level NOAEL of PX-200 for the parent P males and for the offspring F1 generation was considered to be equal to or greater than 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development by repeated oral gavage daily administration to Wistar rats at 50, 250 and 1000 mg/kg bw/day, 4 mL/kg in peanut oil, compared to control animals,treated with the vehicle only.As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to Day 4 post-partum associated with administration of repeated doses. Twelve male andtwelvefemale Wistar rats/group were treated according to the followingExperimental Design:

 

Group no./ Designation	Dose Level (mg/kg bw/day)	Concentration  (mg/mL)	Dose volume (mL/kg)	Animal Numbers
				Male	Female
1/Control	0	0	4	1001-1012	1501-1512
2/Low dose	50	12.5		2001-2012	2501-2512
3/Mid dose	250	62.5		3001-3012	3501-3512
4/High dose	1000	250		4001-4012	4501-4512

 Dose formulation preparation was conducted for use within 1 day at room temperature or 5 days when stored refrigerated at 2-8°C, according to the stability assessment on CiToxLAB study code 10/235-316AN. Analysis of test item formulations for concentration and/or homogeneity was performed at CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations during the first and last week and midway (29 November 2011, 21 December 2011 and 09 January 2012), and one sample was taken from the Group 1 (control) solution for concentration measurements.

No test item was identified in the control samples. The test item formulations appeared homogenous by visual inspection and had actual concentrations of 99-103% of the nominal concentrations. These results were considered within the acceptance criteria of 100±10% and suitable for the study purposes.

Adult animals were inspected for signs of morbidity and mortality twice daily. Clinical observations were performed once daily, with detailed examination performed weekly. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partum periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly.

After delivery, all pups from each litter were counted, sex established, weighed on post-natal days PND 0 and 4, offspring evaluated for presence of stillbirths, live births, runts (pups that are significantly smaller than normal pups) or any gross abnormalities, then examined clinically at least daily. 

Gross necropsy of the adult parental animals was conducted at the end of the treatment period and weights of standard list of organs were measured. Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities.

Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups, found dead female 4503, and any macroscopic findings (abnormalities) observed. Special attention was paid to evaluation of the 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.

There were no test item-related clinical signs or unscheduled mortality recorded during the study.

One female was found dead on Day 11, before the onset of mating, due a dosing accident, unrelated to the test item. As of Day 8, prior to its death, clinical signs including red liquid from the nostrils and hunched back position were noted, followed by decreases in activity on Day 10, then death on Day 11. At necropsy, oesophagus perforation confirmed the dosing accident, associated with white, creamy material in the thoracic cavity, dark red discoloration noted in the lungs and thickened rough surface in the pericardium and pleura. The cause of death was not ascribed to a test item-related effect, but considered due to the moderate, mixed cellular inflammation in the pleura, pericardium and oesophagus caused by the perforation (misgavage) of the oesophagus.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development by repeated oral gavage daily administration to Wistar rats at 50, 250 and 1000 mg/kg bw/day, 4 mL/kg in peanut oil, compared to control animals,treated with the vehicle only.As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to Day 4 post-partum associated with administration of repeated doses. Twelve male andtwelvefemale Wistar rats/group were treated according to the following Experimental Design:

 

Group no./ Designation	Dose Level (mg/kg bw/day)	Concentration  (mg/mL)	Dose volume (mL/kg)	Animal Numbers
				Male	Female
1/Control	0	0	4	1001-1012	1501-1512
2/Low dose	50	12.5		2001-2012	2501-2512
3/Mid dose	250	62.5		3001-3012	3501-3512
4/High dose	1000	250		4001-4012	4501-4512

 

Dose formulation preparation was conducted for use within 1 day at room temperature or 5 days when stored refrigerated at 2-8°C, according to the stability assessment on CiToxLAB study code 10/235-316AN. Analysis of test item formulations for concentration and/or homogeneity was performed at CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations during the first and last week and midway (29 November 2011, 21 December 2011 and 09 January 2012), and one sample was taken from the Group 1 (control) solution for concentration measurements.

No test item was identified in the control samples. The test item formulations appeared homogenous by visual inspection and had actual concentrations of 99-103% of the nominal concentrations. These results were considered within the acceptance criteria of 100±10% and suitable for the study purposes.

Adult animals were inspected for signs of morbidity and mortality twice daily. Clinical observations were performed once daily, with detailed examination performed weekly. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partum periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly.

After delivery, all pups from each litter were counted, sex established, weighed on post-natal days PND 0 and 4, offspring evaluated for presence of stillbirths, live births, runts (pups that are significantly smaller than normal pups) or any gross abnormalities, then examined clinically at least daily. 

Gross necropsy of the adult parental animals was conducted at the end of the treatment period and weights of standard list of organs were measured. Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities.

Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups, found dead female 4503, and any macroscopic findings (abnormalities) observed. Special attention was paid to evaluation of the 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.

There were no test item-related clinical signs or unscheduled mortality recorded during the study.

One female was found dead on Day 11, before the onset of mating, due a dosing accident, unrelated to the test item. As of Day 8, prior to its death, clinical signs including red liquid from the nostrils and hunched back position were noted, followed by decreases in activity on Day 10, then death on Day 11. At necropsy, oesophagus perforation confirmed the dosing accident, associated with white, creamy material in the thoracic cavity, dark red discoloration noted in the lungs and thickened rough surface in the pericardium and pleura. The cause of death was not ascribed to a test item-related effect, but considered due to the moderate, mixed cellular inflammation in the pleura, pericardium and oesophagus caused by the perforation (misgavage) of the oesophagus.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The no observed effect level NOEL and no observed adverse effect level NOAEL of PX-200 for the parent P males and for the offspring F1 generation was considered to be equal to or greater than 1000 mg/kg bw/day.