Butyl (dialkyloxy(dibutoxyphosphoryloxy))titanium(trialkyloxy)titanium phosphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29.02.2016-07.11.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted: 28 July 2015

Deviations:

yes

Remarks:

All changes, were considered to have no impact on the toxicological results of this study. As no amendment or formal change control procedure was initiated, they were each filed as a deviation, but classified as non-critical.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butyl (dialkyloxy(dibutoxyphosphoryloxy))titanium(trialkyloxy)titanium phosphate

EC Number:

401-100-0

EC Name:

Butyl (dialkyloxy(dibutoxyphosphoryloxy))titanium(trialkyloxy)titanium phosphate

Cas Number:

109037-78-7

Molecular formula:

not applicable

IUPAC Name:

dititanium(4+) triethanolate bis(propan-1-olate) dibutyl phosphate butyl phosphate

Test material form:

liquid

Details on test material:

The test material is a clear, yellow liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han (IGS)

Details on species / strain selection:

Rattus norvegicus

Sex:

male/female

Details on test animals or test system and environmental conditions:

Clean conventional housing: airing with approx. 10 air changes per hour, room climate 22 ± 3°C, relative humidity at 30-70%, (actual relative humidity was not altered due to meteorological circumstances), artificial lighting 12 h light/12 h dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item Printcat 508 was administered daily by oral gavage to 12 male and 12 female rats per dose group throughout the in-life phase of this study (see table 2). The test item was administered at three graduated doses in an application volume of 5 ml per kg body weight. The individual dose volumes were calculated from the latest actual animal body weight data. 24 animals (12 males / 12 females) received pure corn oil as vehicle control via the same route.

Details on mating procedure:

During acclimatisation, pre-exposure and pre-mating, male rats were caged in groups of three animals per cage; females were housed individually. Throughout the mating period, animals were kept in pairs of one female and one male rat. The female was placed with the same male until pregnancy occured or two weeks had elapsed. In case a male died during the mating period or successful mating was not be detectable after 14 days, re-mating of females with proven males (sires) of the same group was considered. After the mating period, male rats were housed in groups of three originally formed during pre-mating phase; female rats were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Since direct quantification of the intact chemical Printcat 508-complex was not feasible, Titanium (Ti), as a distinct component of the chemical Printcat 508-complex, was used as the analytical marker for a quantitative re-analysis of the test item dissolved in corn oil. A preparative
procedure had to be validated that causes a complete decomposition of Ti from the chemical Printcat 508-complex as well as from the corn oil matrix. The subsequent fraction-analysis of the released Ti should be carried out via Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES). As part of the method validation, two different protocols for sample preparation (protocols for dry ashing and wet ashing preparations) were carried out in a preliminary study. For either protocol tested, the study results returned a recovery rate below 5% for the released Ti, given that the nominal Ti-concentration in the utilized test item-solution of 1,2 mg/L was applied as the basis for the calculations of the test item-recovery rates. A matrix effect of the dissolvent in combination with the low amount of bound Ti within the chemical Printcat 508-complex could be considered a cause for the low recovery rates. In due consideration of the matrix effect caused by the dissolvent, the determination of the test item in corn oil via measurement of released Titanium per ICP-OES is considered not feasible and for this reason a test item re-analysis was not performed for this study.

Duration of treatment / exposure:

Males were dosed daily for 42 days to 46 days, including the day before the scheduled termination of the in-life phase. This included two weeks of dosing prior to mating and continued throughout the mating period until approximately two weeks post-mating.
Females were dosed two weeks prior to mating, covering at least two complete oestrous cycles, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation and pre-dosing estrus-cycle evaluation depended on the female performance (14 days pre-mating, up to 14 days until mating, an average of 22 days of gestation, and a minimum of 13 days of lactation) and was at least 63 days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

160 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

Not set

Examinations

Parental animals: Observations and examinations:

Cage-side observations to detect signs of illness or reactions to treatment, moribund animals or fatalities were conducted as scheduled in chapter 3.2. Data were recorded within the raw data. For the adult animals, individual body weight, individual/group food and individual/group
water consumption was monitored as scheduled in chapter 3.2. During pregnancy, females were weighed individually on days 0,7,14 and 20 and within 24 hours of parturition (at birth: day 0 or 1 post partum) and on day 4, 9 and on day of scheduled necropsy (at least on day
13 post-partum).

Oestrous cyclicity (parental animals):

Yes

Sperm parameters (parental animals):

Not included

Litter observations:

Each litter was examined as soon as possible after delivery to establish parameters as follow: numbers, sex and presence of gross abnormalities were evaluated for pups, stillbirth, live birth and runts.

Postmortem examinations (parental animals):

All adult animals were sacrificed humanely by asphyxiation in a CO2 atmosphere and were examined macroscopically. All occurring lesions were recorded on checklists for each individual animal. Special attention was paid to the organs of the reproductive system. The number of implantation sides was recorded. The testes, epididymides and the LABC muscle, the cowper’s glands and the glans penis of all male adult animals were surgically extracted and weighed as soon as possible after dissection. Paired organs were weighed individually. The ovaries, testes, epididymides, accessory sex organs (cervix, prostate etc.) and all other organs showing macroscopic lesions of all adult animals were
preserved in the appropriate fixative. Adult animals: Terminal procedures, tissue preservation, organ weights and implantation sites.

Postmortem examinations (offspring):

Dead pups and pups humanely killed on day 13 post partum, or shortly thereafter, were examined externally for gross abnormalities. External reproductive genitals were examined for signs of altered development. Immediately after death, the thyroid gland from one male and one female pup per litter were preserved.

Statistics:

Spread sheet calculations were performed using Microsoft® Excel® 2011 for Mac. Data on body weight were recorded for each individual animal. Food- and water consumption were documented sorted by experimental groups and sex. For each experimental group, means and standard deviations were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animal behavioral signs of wiping of nose and mouth through cage bedding, salivation before and after dose application were especially observed in all test item-treated male animal groups, but the findings were predominantly observed at the high dose. Although the observed behavioral signs were probably related to the test item, they were considered to be of minor toxicological importance. No adverse clinical signs were observed during the course of the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On premating day 2, one female of the low dose group (E79/0) was found dead. Necropsy did not identify a plausible cause for the death. Based on the animal’s behavior immediately after the last dose administration [animal lay outstretched in cage, behaved lethargic), it is assumed that a gavage error was most likely the cause of death. While there were no necropsy findings to support this conclusion, and the lung volume was not checked for remnants of the test item at necropsy, this effect was not considered test item-related.
Additional animals were found dead on pre-mating day 3 [one female animal of the low dose group (E104/0)], pre-mating day 5 [one female animal of the high dose group (E108/0)], day 14 of the pre-mating phase [one male animal of the low dose group (E62/1)], and day 20 of gestation [one female animal of the medium dose group (E76/0)]. In all cases, test item was found inside the lungs at necropsy, so that aspiration caused by a failed administration was regarded as the most plausible cause for the death of the animals. Thus, these deaths were considered not test item-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Over the course of the study no statistical differences in the mean body weights of female test groups and the respective control group detected. Correspondingly, the mean body weight gain was comparable between all female groups either treated with the test item or the vehicle control item. All values were within normal range for female rats of this strain and age. Occasional differences especially towards the end of the gestation phase were assumed to be within the physiological variation caused by the pregnancy status of the animals.
For male animals, no statistical differences in mean body weights were observed between the test groups and the respective control group over the course of the study. Correspondingly, the mean body weight gain was comparable between all male animal groups either treated with the test item or the vehicle control item. All values were within normal range for rats of this strain and age.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In summary, the monitored time intervals did not reveal any changes of toxicological relevance in regard to food consumption between the animal test groups and their respective vehicle control groups throughout male and female in-life phases. Some variances were observed for the female animal groups, which could be associated with the specific animal condition during phases of late pregnancy and subsequent lactation.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

In summary: the majority of monitored intervals revealed a test item-related slight increase in water consumption for the female high and medium dose groups, when compared to the respective vehicle control group. For the male dose groups no test item related-effect could be observed in this respect. The observed test item–related differences in water consumption in the majority of monitored time intervals could be regarded as within the normal range for female animals of this strain and age and were therefore not considered adverse. The differences in female water consumption observed between late pregnancy/lactation and the earlier in life-phases resulted from the specific physiological condition of the animals and therefore considered as not adverse or even toxicologically relevant.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In comparison to the respective vehicle controls, no difference in total T4-hormone content in blood plasma was detected for the sires and the day 13 pp pups in a t-test analysis. In summary: no test item-related change of total T4-hormone content in blood plasma could be detected in the experimental animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

With the exception of one female animal (high dose), only males and sires showed particular animal behavior, predominately at the high dose. The role of the test item on these effects could not be fully excluded. These observed effects were considered not adverse as they were mild in severity and had a minor impact on the experimental animals during the course of the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

According to guideline OECD421, tissue samples from a selective panel of reproductive organs and macroscopic lesions as well as vaginal smears prepared at necropsy from dams and sires of the experimental high dose group and respective vehicle control group were correlated and conclusively analyzed by the histopathologist. The histopathological analysis (on macroscopic and microscopic level) revealed no evidence for a test item-related effect on the reproductive organs from experimental adult animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The daily monitoring of the animal’s estrus cycles revealed a regular cycle for each experimental female at the start of the mating phase, hence each female was considered for participation in the subsequent mating.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Germ cells in the testis were evaluated by identifying tubules with respect to their stage in the spermatogenic cycle. Neither germ cell depletion nor spermatid retention were found. The population of Sertoli cells was normal and interstitial Leydig cells did neither show atrophy or hyperplasia. Epididymides did not show desquamated germ cells, cell debris or reduced number of sperm.
Unilateral focal sperm granuloma in the epididymis was found in one animal treated with 1000 mg/kg (animal no. E60/0), but is not considered test item-related, because it is a common spontaneous finding in Wistar rats that may develop as a consequence of congenital ductal aplasia.

Reproductive performance:

no effects observed

Description (incidence and severity):

In regard to evaluated parameters “numbers of pups, abnormal pups and loss of offspring” no statistical significant differences between the test groups and the respective vehicle control group were noted.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 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

water consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Within the litter (11 pups in total) of the high dose group-animal E73/0 and within the litter (16 pups in total) of the medium dose group-animal E88/0 one stillborn pup each was found. Within the litter (8 pups in total) of the low dose group-animal E80/0 two stillborn pups were found and within the litter (13 pups in total) of the vehicle group-animal E93/0, one stillborn pub was identified.
The examination of each stillborn`s body did not reveal any signs of deformation or developmental delays. The distribution of identified stillborns within the experimental groups did not correlate in a dose-dependent manner. Therefore a test item-related effect could be excluded for each particular incident with high probability.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose group, one animal (E84/0) gave birth to 13 pups, from which one was identified as physically smaller when compared to its siblings [body weights: 3,6g / 6,2 g (small pup / mean pup weight/litter)] and to pups of the vehicle group at necropsy. Besides considerably smaller body size, the runt showed no other signs of developmental delay or signs of deformation until end of the in-life phase.
In the medium dose group, one animal (E87/0) gave birth to 13 pups from which one was identified as physically smaller when compared to its siblings on day d0 pp. However, the particular pup went missing the next day. An apparent cause for the pup’s death was not identified.
Another animal of the medium dose group (E113/0) gave birth to 2 pups of which one pup was considerably smaller in size, when compared to its normal sized sibling at day of actual birth (d0 pp) or to normal sized pups of the vehicle group at necropsy. The smaller pup showed no other signs of developmental delay or signs of deformation until end of the in-life phase. The normal sized pup vanished at d1 pp.
One animal of the vehicle control group (E107/0) gave birth to 5 pups of which one pup was identified as a runt when compared to its siblings. The particular pup showed no other signs of developmental delay or signs of deformation until end of its in-life phase.
Individual pups from litters of all dose groups and the vehicle group did not have their eyes open on day 13 post partum and retroorbital blood sampling could not be performed. As the pups from litters of all experimental groups had at latest their eyes open at day 14 post partum, no difference in pup development could be identified between the pups of the dose groups and the pups of the vehicle group.
However, in some animal litters, individual pups with delayed body growth were identified along with other siblings, which were regarded as normal in size and appearance. The occurrence of these particular pups within litters of the different experimental groups did not reveal a dose-dependent correlation. Therefore a test item-related effect was considered as most unlikely.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The careful examination of d13 pp pups did not return any signs of gross abnormalities either in terms of general toxicity or in terms of alterations or developmental delays of organs within the reproductive system. According to the macroscopic results a test item-related effect on the organs relevant within the reproduction system can be excluded with high probability.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

In regard to evaluated parameters “sex ratio, anogenital distance (AGD) and nipple retention” no statistical significant differences were detected between the test groups and the respective control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

gross pathology

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

A daily oral administration of the test item Printcat 508 to male and female Wistar rats up to 1000 mg/kg/day in current reproduction/developmental screening did not lead to adverse toxicity to P0 animals and neither an effect on the reproduction performanc as well as development of F1 pups. Therefore it is concluded the NOAEL for parental systemic and reproduction toxicity, as well as fetal development toxicity is 1000 mg/kg/day.

Executive summary:

A daily oral administration of the test item Printcat 508 to male and female Wistar rats at dose levels of 1000 mg, 400 mg and 160 mg/kg body weight over a time period of 51 to 63 days for males and 42 to 46 days for females resulted in some minor animal behavioral changes in male animals and some minor changes in the water consumption of females. Neither were considered adverse nor toxic. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the subchronic toxicity was set to 1000 mg/kg body weight. Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of Printcat 508 under these study conditions was estimated to be 1000 mg/kg body weight for the female and male animals.