Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 421 there were no direct adverse effects on reproduction observed. In terms of reproduction, there was no adverse effect on mating or pregnancy rate at dosages up to 60/90/60 mg/kg bw/day. 

Lower numbers of implantations were apparent for all dosage groups but the lack of any dosage relationship indicated that this finding was unrelated to treatment. 

At 60/90/60 mg/kg bw/day, four females showed high incidences of post-implantation loss and, this combined with the high incidence of post-natal losses, probably precludes this from being viewed as a No Observed Adverse Effect Level (NOEL) for reproduction. At 30 mg/kg bw/day, the two females that showed total litter loss post partum. These effects are suspected as being due to adult toxicity, appear to preclude the high and mid dose dosage from being a NOEL for reproduction. However, at 30 mg/kg bw/d next to the litter loss there was no other apparent effect on reproduction and therefore this dosage may be classified as a NOAEL for reproduction. The No Observed Effect Level (NOEL) for the reproduction in this study was considered to be 15 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 21 December 2016 Experimental Completion Date: 14 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification : Dichlorobis(n-cyclopentadienyl)titanium
(CAS# 1271-19-8)
Physical State/Appearance : Red powder
Chemical Name : bis(cyclopentadienyl) titanium dichloride
Purity : 99.3%
Batch Number : 1504510829
Date Received : 21 June 2016
Storage Conditions : Ambient conditions, in the dark; used/formulated in light
Expiry Date : 03 April 2019
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate guideline authorities.
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for twenty six days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 271 to 347g and were approximately eleven weeks old and the females weighed 184 to 247g, and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Corn oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least eleven days when stored refrigerated (approximately 4°C) in the dark. Formulations were initially prepared on a daily basis until this stability was confirmed and then prepared on an approximately weekly basis and stored at approximately 4 °C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of Dichlorobis(n-cyclopentadienyl)titanium at Envigo Research Limited, Shardlow, UK, Analytical Services on four occasions during the study. The results indicate that the prepared formulations were within 99-108% of the nominal concentration confirming the suitability of the formulation procedure for the purpose of this study.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item described in the main part of this study was also used as the analytical standard.

Preparation of Claibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot of test item was exactly weighed into a volumetric flask and brought to volume with dilution solvent. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL. Standard solutions contained the equivalent amount of vehicle to that of the relevant samples.

On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis sequence as a minimum.

To assess the calibration range of the method, a range of standard solutins were prepared in dilution solvent and vehicle covering the contration range 0.0496 mg/mL to 0.1848 mg/mL.

Preparation of Test Samples
The formulations received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent, which was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentrations.

Preparation of Accuracy and Precision Samples
Samples of Corn Oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis.

The concentration of Test Item in the final solution was quantified by HPLC using UV detection.

Instrumentation Parameters
HPLC: AgilentTechnologies 1200, incorporatng autosampler and workstation
Column: Luna 5 µ silica (250 x 4.6 mm id) at 30°C
Mobile phase: Tetrahydrofuran
Flow-rate: 1 mL/min
UV detector wavelength: 254 nm
Injection volume: 10 µL
Retention time: ~3.4 mins
Duration of treatment / exposure:
approximately six weeks for males and a two week pre-pairing phase, pairing, gestation and lactation to Day 14 for females
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
iii. For the 14 days prior to pairing pre-pairing vaginal smears were performed and assessed for females.
iv.On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vii. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.
viii. The male dose groups were killed and examined macroscopically on Day 44 or 45.
ix. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All offspring were killed and examined externally, where external observations were detected an internal necropsy was performed.
x. All females were killed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also killed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all animals at termination for possible thyroid hormone analysis.
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
increased to 90 mg/kg bw/day from Day 10 and subsequently lowered back to 60 mg/kg bw/day for females only from Day 44 (around the time females were littering).
No. of animals per sex per dose:
12 males/ 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected in collaboration with the Sponsor Representative and were based on available toxicity data including preliminary data from a fourteen day dose range-finding toxicity study in the rat (Envigo Study Number YY06FT). In the range-finding study, dose levels of 30, 60 or 125 mg/kg bw/day resulted in lower overall body weight gains in a dose-related manner with the effect appearing to be more pronounced in males. This was associated with slightly lower overall food consumption in animals of either sex treated with 125 or 60 mg/kg bw/day with an increase in overall water consumption also being evident for the high dose males. At necropsy, macroscopic findings were confined to females from the 125 mg/kg bw/day dose group where 2/3 animals showed red discoloration of the glandular region in the stomach. A high dose level of 60 mg/kg bw/day was therefore initially considered to be suitable for investigation in this OECD 421 together with 15 and 30 mg/kg bw/day as the low and intermediate dose levels, respectively. Following a review of the available in-life data from the first week of dosing, the high dosage was increased to 90 mg/kg bw/day from 25 January 2017 (Day 10). Following the premature termination of two high dosage females and increased offspring mortality for a further high dosage litter, the high dosage for females was lowered to 60 mg/kg bw/day from 28 February 2017 (Day 44).
Positive control:
Not applicable
Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period for the periods covering post partum Days 1-4, 4-7 and 7-14.

Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Testes weight and histopathology
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.
Postmortem examinations (parental animals):
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were killed via carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy were killed around the same time as littering females.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination; where external observations were detected an internal examination was performed.

Thyroid Hormone Assessment
Where possible, blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormone. Blood samples to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60 ºC for possible evaluation of thyroid hormones. Samples were taken as follows:

Where possible from each litter, serum samples were taken from two randomly allocated offspring on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.

Where possible from each litter, serum samples were taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum.

Serum and plasma samples were taken from all adult males and all adult females at termination.

All serum samples were shipped to the test site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) for serum analysis, frozen, packed in dry ice. The serum from adult males and Day 13 offspring were analyzed for Thyroxine (T4) under the supervision of the Prinicpal Investigator (H Bose) All plasma samples were retained at the Test Facility.

Organ Weights
The epididymides, testes, seminal vesicles (with coagulation gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed post-fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Cowpers glands
Pituitary
Epididymides ♦
Prostate
Glans penis
Seminal vesicles (with coagulating gland)
Gross lesions
Testes ♦
LABC (levator ani-bulbocavernous) muscle
Thyroid/parathyroid
Ovaries
Uterus/Cervix (and oviducts)
Mammary gland
Vagina

All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing. The tissues from control and high dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from these male animals were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
4.5.3 Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by Vasanthi Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.
Postmortem examinations (offspring):
Thyroid Hormone Assessment
Where possible from each litter, serum samples were taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum.
Statistics:
Please refer to "Any other information of materials and methods including tables"
Reproductive indices:
Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live off-spring / Number of pregnant females) x 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).

i. Post-Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:

Post–implantation loss (%) = ((Number of implantation sites - Total number of off-spring born) / Number of implantation sites) x100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Viability Index 1 (%) = (Nukber of offspring alive on Day 4 / Number of offpsring alive on Day 1) x 100

Viability Index 2 (%) = (Number of offspring alive on Day 7/13 / Number of offspring alive on Day 4) x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula:

(Number of male offpsring / Total number of offspring) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed for surviving animals that indicated any adverse effect of treatment for either sex at 15 or 30 mg/kg bw/day, males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.

Increased post-dosing salivation was observed for the majority of males and, at a lower incidence, the majority of females at 60/90/60 mg/kg bw/day. This finding is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect unpalatability or slight irritancy of the test item formulation, rather than any systemic effect of treatment.

Noisy respiration was occasionally observed for isolated treated animals on sporadic occasions during the study. However, the distribution and incidence of this finding indicated that it was most probably associated with occasional difficulties in dosing animals rather than any indication of test item toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Following gestation, two females treated with 60/90 mg/kg bw/day showed a deterioration in clinical condition around the time of parturition that resulted in their early termination and necessitating the lowering of the high dosage for females to 60 mg/kg bw/day. At necropsy, both animals showed red/black coloured stomach contents (this may reflect consumption of the placentae following delivery of the litter), sloughing of the stomach and red patches on the glandular region of the stomach. Other stomach findings included distension, raised limiting ridge and raised white patches on the non-glandular region. Additionally, both animals showed black contents in the cecum, which appeared small and one animal also showed black content in the colon.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment of females at dosages of 15, 30 or 60/90 mg/kg bw/day during the two week pairing phase was not associated with any effects on body weight or food consumption that were considered to be related to treatment. Statistically significant lower body weight gain was apparent at 30 and 90 mg/kg bw/day during Week 1, compared to control, but the extent of these differences considering the age of the animals and the lack of dosage relationship indicated that these findings were incidental and unrelated to treatment.
At 60/90 mg/kg bw/day, body weight gain and food consumption of females during gestation were statistically significantly lower than control. During late gestation, these values, particularly body weight gain, may been influenced by lower in utero litter size, however the lower female body weight observed at the start of lactation would tend to indicate an underlying effect of treatment on the females during late gestation.


Body weight gain and food consumption of females during lactation was lower than control. The differences in body weight gain did not attain statistical significance compared to control, but given the lower litter size, and therefore lower demand from the suckling litter, it may have been expected for body weight performance to show greater parity with control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption did attain statistical significance when compared with control on Days 4-7 and 7-14 of lactation, and these differences may well be attributable to lower demand from the suckling litter.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 60/90 mg/kg bw/day, food conversion efficiency for males appeared inferior to control during the second week of treatment (pre-pairing) and during the post-pairing phase of the study.

At 15 and 30 mg/kg bw/day, values for food conversion efficiency for males also tended to be lower than control, however the magnitude of these differences and the lack of any consistent dosage relationship did not indicate any obvious effect of treatment.

Intergroup differences in food conversion efficiency for females during the pre-pairing phase of the study did not indicate any effect of treatment at 15, 30 or 60/90 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any effect of treatment for either sex throughout the study.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
For males at 90 mg/kg bw/day, lower absolute and body weight relative prostate weights attained statistically significance compared to control.

There were no statistically significant differences in male reproductive organ weights apparent at 15 or 30 mg/kg bw/day.

There were no statistically significant differences from control for thyroid weight of either sex at 15 or 30 mg/kg bw/day, for males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any effect of treatment on offspring development at 15, 30 or 60/90/60 mg/kg bw/day.
At 60/90/60 mg/kg bw/day, it was noted that a number of decedent offspring and offspring killed at Day 4 of age showed no milk in the stomach, a finding consistent with the clinical signs observed for the young offspring at this dosage.
Adults
Neither the type, incidence or distribution of necropsy finding for surviving animals indicated any obvious effect of treatment for either sex at 15 or 30 mg/kg bw/day, for males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of reproductive tissues (testes, epididymides and ovaries) from the control and high dosage animals (60/90 mg/kg bw/day for males and 60/90/60 mg/kg bw/day for females) did not reveal any findings considered to be related to treatment with the test item. In particular, no consistent treatment-related pathologic findings in the testes following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or in the ovaries following the evaluation of the follicles and corpora lutea.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males did not identify any effect of treatment or indication of endocrine disruption at 15, 30 or 60/90 mg/kg bw/day.
Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 15, 30 or 60/90/60 mg/kg bw/day.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In terms of reproduction, there was no adverse effect on mating or pregnancy rate at dosages up to 60/90/60 mg/kg bw/day. Lower numbers of implantations were apparent for all dosage groups but the lack of any dosage relationship indicated that this finding was unrelated to treatment. At 60/90/60 mg/kg bw/day, four females showed high incidences of post-implantation loss and, this combined with the high incidence of post-natal losses, probably precludes this from being viewed as a No Observed Adverse Effect Level (NOEL) for reproduction. At 30 mg/kg bw/day, the two females that showed total litter loss post partum, although these deaths are suspected as being due to adult toxicity, appear to preclude this dosage from being a NOEL for reproduction. However, these litters aside, there was no other apparent effect on reproduction and therefore this dosage may be classified as a NOAEL for reproduction. The No Observed Effect Level (NOEL) for the reproduction in this study was considered to be 15 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Remarks:
Reproduction
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 60/90/60 and, to a lesser extent 30 mg/kg bw/day, the incidence of offspring clinical signs from birth to Day 4 of age was higher than control. The types of clinical signs observed were unremarkable and did not indicate any obvious developmental effect on the offspring at these dosages. It was noted that some/all offspring from four litters at 60/90 mg/kg bw/day showed no indication of milk in the stomach; all offspring from three of these litters failed to survive to Day 4 and the other litter showed poor offspring survival. There were no incidences of this finding at 30 mg/kg bw/day. The type and incidence of offspring clinical signs at both dosages after Day 4 of age did not indicate any effect of maternal treatment.
The type and incidence of offspring clinical signs at 15 mg/kg bw/day did not indicate any effect of maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
. It was noted that some/all offspring from four litters at 60/90 mg/kg bw/day showed no indication of milk in the stomach; all offspring from three of these litters failed to survive to Day 4 and the other litter showed poor offspring survival.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 60/90/60 mg/kg bw/day, mean body weight of the offspring on Day 1 and subsequent mean body weight gain to termination on Day 13 of age was slightly lower than control, but differences failed to attain statistical significance. Litter weights were statistically significantly lower than control throughout but this principally reflected the lower litter sizes at this dosage compared to control.
There was no effect of maternal treatment on offspring body weight on Day 1 or subsequent body weight gain to termination on Day 13 apparent at 15 or 30 mg/kg bw/day. At both dosages, litter weights were lower than control but these differences reflected the differences in litter sizes at these dosages compared to control, as mean offspring weights were higher than control from Day 1 to termination.
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):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any effect of treatment on offspring development at 15, 30 or 60/90/60 mg/kg bw/day.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Treatment of males at 60/90 mg/kg bw/day was associated with lower body weight gain from the second week of treatment (treatment was increased to 90 mg/kg bw/day on Day 10) resulting in lower body weight and overall body weight gain at termination. These lower gains appear to reflect inferior food conversion efficiency, compared with control, as there was no obvious effect on male food consumption during the study. There were no clinical signs or macroscopic necropsy findings that were considered to indicate a systemic effect of treatment. Mean absolute and body weight-relative prostate weights were lower than control but all individual values were within the historical control range. There were no treatment related findings apparent for the testes or epididymides, which were both examined microscopically, so this isolated finding for the prostate was considered most likely to be unrelated to treatment and of no toxicological significance.

Treatment of males at 15 and 30 mg/kg bw/day was well tolerated with no indication of systemic toxicity being apparent.  

Treatment of females at dosages of 15, 30 or 60/90 mg/kg bw/day during the two week pairing phase was not associated with any effects on body weight or food consumption that were considered to be related to treatment. Statistically significant lower body weight gain was apparent at 30 and 90 mg/kg bw/day during Week 1, compared to control, but the extent of these differences considering the age of the animals and the lack of dosage relationship indicated that these findings were incidental and unrelated to treatment.  

At 60/90 mg/kg bw/day, body weight gain and food consumption of females during gestation were statistically significantly lower than control. During late gestation, these values, particularly body weight gain, may been influenced by lowerin uterolitter size, however the lower female body weight observed at the start of lactation would tend to indicate an underlying effect of treatment on the females during late gestation.

Following gestation, two females treated with 60/90 mg/kg bw/day showed a deterioration in clinical condition around the time of parturition that resulted in their early termination and necessitating the lowering of the high dosage for females to 60 mg/kg bw/day. At necropsy, both animals showed red/black coloured stomach contents (this may reflect consumption of the placentae following delivery of the litter), sloughing of the stomach and red patches on the glandular region of the stomach. Other stomach findings included distension, raised limiting ridge and raised white patches on the non-glandular region. Additionally, both animals showed black contents in the cecum, which appeared small and one animal also showed black content in the colon.

At 60/90/60 mg/kg bw/day, the distribution of gestation lengths attained statistical significance compared with control but, as this distribution was similar to that at 15 mg/kg bw/day, an association with treatment appears unlikely. One decedent female killedin extremisaround the time of parturition did show a notably long gestation period (24 days) but it was unclear whether this extended gestation period was caused by the poor clinical condition of the female or was a contributing factor in the decline in the clinical condition of the animal. There was no obvious association between gestation length and the incidence of decedent females/females showing total litter losspost partum.               

Body weight gain and food consumption of females during lactation was lower than control. The differences in body weight gain did not attain statistical significance compared to control, but given the lower litter size, and therefore lower demand from the suckling litter, it may have been expected for body weight performance to show greater parity with control. Food consumption did attain statistical significance when compared with control on Days 4-7 and 7-14 of lactation, and these differences may well be attributable to lower demand from the suckling litter. 

At 60/90/60 mg/kg bw/day, the mean number of implantations was slightly lower than control but intergroup differences indicated that this finding was incidental and unrelated to treatment. Subsequently, the mean total number of offspring born was also slightly lower than control, however, while this finding is consistent with the previous lower implantation count, four females showed relatively high incidences of post-implantation losses. Three of these females (one of whom was later killedin extremis) subsequently showed total litter losspost partum. It should be noted that post-implantation loss may include offspring that were actually born but not observed before being cannibalized by the mother. Live litter size on Day 1 was clearly lower than control due to inferior offspring survival after parturition, as indicated by inferior mean live birth index compared to control. Subsequent offspring survival for litters surviving to termination was also inferior to control to Day 4 of age. These findings have to be considered in the context of four females (one of whom was subsequently killedin extremis) that showed total litter losspost partumduring early lactation. Subsequent offspring survival from Day 4 to termination on Day 13 was similar to control and appeared unaffected by maternal treatment.

At 60/90/60 mg/kg bw/day, mean body weight of the offspring on Day 1 and subsequent mean body weight gain to termination on Day 13 of age was only slightly lower than control, with differences failing to attain statistical significance. Considering the smaller size of the litters, it may have been expected for offspring growth to show greater parity with their control counterparts. Litter weights were lower than control throughout lactation and, while this would have been influenced by lower offspring weight gain, it principally reflected the lower litter sizes at this dosage compared to control.     

Despite the higher offspring mortality and lower weight gain at 60/90/60 mg/kg bw/day, clinical signs and necropsy findings for the offspring did not indicate any obvious effect on offspring development. There was no effect of treatment on ano-genital distance for either sex and, although the incidence of offspring clinical signs from birth to Day 4 of age was higher than control, the clinical signs and necropsy findings observed were unremarkable. Offspring from four litters did show no indication of milk in the stomach during this period with all offspring from three litters failing to survive to Day 4 and the remaining litter showing poor offspring survival. A number of decedent offspring or offspring killed at Day 4 of age also showed no milk in the stomach. It is not clear whether this resulted from a failure of the offspring to suckle or low quality/quantity of milk production by the female.     

To a certain extent, the assessment of effects on body weight and food consumption at 30 mg/kg bw/day was complicated by the lower litter size observed at this dosage. Food consumption of females was lower than control throughout gestation, however statistically significant lower body weight gain was only apparent during the last week of gestation, although this did result in lower overall body weight gain. The lower litter size may have influenced both food consumption and, particularly, body weight gain during late gestation. The lower body weight at the start of lactation may indicate a slight underlying effect on the dam at this stage of the study.  

There was considered to be no effect of treatment on body weight gain or food consumption of females during lactation at 30 mg/kg bw/day. Although lower food was apparent for females during lactation at this dosage, this was considered to reflect lower litter demand from the smaller litters at this dosage.  

At 30 mg/kg bw/day, the mean number of implantations was slightly lower than control but intergroup differences indicated that this finding was incidental and unrelated to treatment. Mean total number of offspring born was slightly lower than control but was generally consistent with the original difference from control established previously for implantation count, although slightly higher mean implantation loss was apparent. Post-natal survival of the offspring from litters reared to termination on Day 13 was unaffected by maternal treatment, therefore lower mean live litter sizes apparent from Day 1, compared to control, reflected differences in litter size already established at birth. However, although post-natal survival amongst these litters was good, two females at this dosage did show total litter losspost partumand, in view of the higher litter/offspring mortality apparent at 60/90/60 mg/kg bw/day, an association with these litter losses at 30 mg/kg bw/day and treatment is difficult to fully discount.

There was no effect of maternal treatment on offspring body weight on Day 1 or subsequent body weight gain to termination on Day 13 apparent at 30 mg/kg bw/day. Litter weights were lower than control throughout lactation reflecting the lower litter size at this dosage compared to control. The incidence of offspring clinical signs from birth to Day 4 of age was slightly higher than control, but neither the clinical signs or necropsy findings observed at this dosage indicate any obvious developmental effects on the offspring. There were no incidences of no milk in stomach apparent during clinical observations or necropsy examinations.

There was considered to be no effect of treatment on the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 15 mg/kg bw/day. The mean number of implantations was slightly lower than control but intergroup differences indicated that this finding was incidental and unrelated to treatment. 

At 15 mg/kg bw/day, body weight gain of females during the first week of gestation was lower than control but, in the absence of any similar effect during this period at 30 mg/kg bw/day, was considered incidental and unrelated to treatment. This lower initial gain during gestation, in combination with a lower contribution from the gravid uterus due to lower
in uterolitter size, compared to control, resulted in overall weight gain during gestation being lower than control. However, the body weight of the females at the start of lactation did not indicate any underlying effect on the dam at this stage of the study. There was no effect on food consumption of females during gestation at this dosage.

At 15 mg/kg bw/day, body weight gain during lactation appeared unaffected by treatment. Although food consumption during lactation at this dosage was lower than control, this was considered to reflect lower litter demand due to lower litter size at this dosage.

Overall, it was considered that the effects observed for females and their litters during the early post-natal phase of the study at 60/90/60 mg/kg bw/day were the most significant toxicological findings apparent during the study. Although the underlying cause of these effects could not be established, differences from control for body weight and food consumption during gestation, high post-implantation loss for four females, the close proximity of parturition with adverse clinical signs leading to the early termination of two dams and the incidence of offspring without milk, particularly for litters that did not survive the early post-natal period, suggests that the female was unable to cope with the physiological demands around the time of parturition rather than indicating a selective adverse effect on the offspring.  Although the assessment of effects on the adult female is limited within this study design, these findings are considered to indicate an adverse effect on the parental female. At 30 mg/kg bw/day, treatment during the late gestation, parturition and early lactation phases of the study appeared much better tolerated, however, there were still two females that showed total litter losspost partum. It is difficult to fully discount an association with treatment with these litter losses in view of the findings observed at the highest dosage on this study, and therefore they may indicate an under lying effect of treatment on the parental female at this lower dosage. In view of this the No Observed Effect Level (NOEL) for the adult animals in this study was considered to be 15 mg/kg bw/day.

In terms of reproduction, there was no adverse effect on mating or pregnancy rate at dosages up to 60/90/60 mg/kg bw/day. Lower numbers of implantations were apparent for all dosage groups but the lack of any dosage relationship indicated that this finding was unrelated to treatment. At 60/90/60 mg/kg bw/day, four females showed high incidences of post-implantation loss and, this combined with the high incidence of post-natal losses, probably precludes this from being viewed as a No Observed Adverse Effect Level (NOEL) for reproduction. At 30 mg/kg bw/day, the two females that showed total litter losspost partum, although these deaths are suspected as being due to adult toxicity, appear to preclude this dosage from being a NOEL for reproduction. However, these litters aside, there was no other apparent effect on reproduction and therefore this dosage may be classified as a NOAEL for reproduction. The No Observed Effect Level (NOEL) for the reproduction in this study was considered to be 15 mg/kg bw/day.

For the offspring, despite the higher offspring mortality and lower weight gain observed at 60/90/60 mg/kg bw/day, clinical signs and necropsy findings did not indicate any effect on morphological development. Ano-genital distance on Day 1 was unaffected and body weight gain of the offspring was only slightly lower than control. However high mortality of the offspring was observed during until Day 4 of age and while this was considered to reflect maternal toxicity rather than a selective effect on the offspring, the incidence of offspring mortality at this dosage was probably too high to consider this being a NOAEL for the offspring. At 30 mg/kg bw/day, the incidence of offspring mortality was lower, although two total litter losses did occur, and this dosage may represent a NOAEL for the offspring. The No Observed Effect Level (NOEL) for the offspring in this study was considered to be 15 mg/kg bw/day.

Conclusions:
Based on the results of this study, a dosage of 15 mg/kg bw/day Dichlorobis(n-cyclopentadienyl)titanium (CAS# 1271-19-8) is considered to be the No Observed Effect Level (NOEL) for adult toxicity, reproduction and the developing offspring. A dosage of 30 mg/kg bw/day may represent a No Observed Adverse Effect Level (NOAEL) for reproduction and the developing offspring.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females). Dose levels of 15, 30 and 60 mg/kg bw/day were initially employed, with the high dosage being increased to 90 mg/kg bw/day from Day 10 and subsequently lowered back to 60 mg/kg bw/day for females only from Day 44. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil) over the same treatment period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with pregnant females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance on Day 1 and visible nipple count on Day 13 (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all females through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning of the day of termination for all females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues from control and high dose animals was performed.


Results…….

Adult Responses

Mortality

There were two unscheduled deaths on the study, both occurring for females at 60/90 mg/kg bw/day around the time of parturition. 

Female 89 was killed on Day 41 after showing piloerection, lethargy, hunched posture, dehydration and diarrhoea. Necropsy revealed red coloured contents in the stomach which appeared thin and distended and showed sloughing and raised white patches on the non-glandular region and red patches on the glandular region. Additionally, black contents were observed in the colon and cecum and the cecum also appeared small. 

Female 94 was killed on Day 43 of the study after showing piloerection, lethargy, hunched posture, dehydration and staining of the fur and around the eyes and snout. Necropsy revealed pale kidneys and black coloured contents in the stomach which showed sloughing, a raised limiting ridge and red patches on the glandular region. Additionally, black contents were observed in the cecum which appeared small.

Clinical Observations

There were no clinical signs observed for surviving animals that indicated any systemic effect of treatment for either sex at 15, 30 mg/kg bw/day or males at 60/90 mg/kg bw/day and females at 60/90/60 mg/kg bw/day.

Body Weight

At 60/90 mg/kg bw/day, body weight gains of males were generally lower than control from the second week of treatment, resulting in lower body weight and overall body weight gain at the end of the study.

There was no effect of treatment on body weight gain of males at 15 or 30 mg/kg bw/day throughout the study. 

There was no effect of treatment on body weight gain of females during the pre-pairing phase of the study at 15, 30 or 60/90 mg/kg bw/day. 

At 60/90 mg/kg bw/day, body weight gain of females during gestation was lower than control, although the differences from control observed during late gestation were, in part, attributable to a lower contribution from the gravid uterus due to slightly lower litter size at this dosage compared to control.  

At 30 mg/kg bw/day, body weight gain of females during the last week of gestation was slightly lower than control, resulting in lower overall body weight gain at the end of gestation. These differences from control were probably attributable to lower contribution from the gravid uterus due to the slightly lower litter size at this dosage compared to control.   

At 15 mg/kg bw/day, overall body weight gain of females during gestation was lower than control but, was influenced by a lower contribution from the gravid uterus due to the slightly lower litter size at this dosage, compared to control.  

At 60/90/60 mg/kg bw/day, body weight gain of females during lactation was lower than control, despite a probable lower demand on the females from the smaller litters at this dosage compared to their control.

At 15 and 30 mg/kg bw/day, body weight gain during lactation was unaffected by treatment.  

Food Consumption

There was no effect of treatment on food consumption of males throughout the study at 15, 30 or 60/90 mg/kg bw/day.

There was no effect of treatment on food consumption of females during the pre-pairing phase of the study at 15, 30 or 60/90 mg/kg bw/day.

At 30 and 60/90 mg/kg bw/day, food consumption of females was lower than control throughout gestation but, to some extent, may have been influenced by slightly lower litter sizes at these dosages compared to control. At 15 mg/kg bw/day, there was no effect of treatment on food consumption of females during gestation.

At 60/90/60 mg/kg bw/day, food consumption of females was lower than control throughout lactation, but, to some extent, may have been influenced by slightly lower litter sizes at this dosage compared to control.

At 15 and 30 mg/kg bw/day, food intake was slightly lower than control throughout lactation, but differences were probably attributable to lower litter demand due to lower litter size at these dosages, compared to control.  

Food Conversion Efficiency

At 60/90 mg/kg bw/day, food conversion efficiency for males appeared inferior to control during the second week of treatment (pre-pairing) and during the post-pairing phase of the study. At 15 and 30 mg/kg bw/day, food conversion efficiency for males appeared unaffected by treatment.

Food conversion efficiency for females during the pre-pairing phase of the study was unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day. 

Water Consumption

Visual inspection of water bottles did not indicate any effect of treatment for either sex throughout the study.      


Reproductive Performance

Estrous Cycle

Estrous cycles during the pre-pairing phase of the study were unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Mating

Mating performance pre-coital interval was unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Fertility

Fertility, as assessed by the number of females that achieved pregnancy, was unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Gestation Length

Gestation length appeared unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

At 60/90/60 mg/kg bw/day, the mean number of offspring born was slightly lower than control but consistent with previous implantations count. Four females showed relatively high incidences of post-implantation losses and three of these females (including one female which was later killedin extremis) subsequently showed total litter losspost partum. Mean live litter size on Day 1 was lower than control due to inferior offspring survival, indicated by lower mean live birth index. Subsequent offspring survival was also inferior to control to Day 4 of age, indicated by lower Day 4 viability index. The lower live litter size on Days 1 and 4 have to be considered in the context of four other females (one of whom was subsequently killedin extremis) that showed total litter losspost partumshortly after parturition. Subsequent offspring survival from Day 4 for surviving litters was unaffected by maternal treatment, therefore the lower litter size on Day 7 and 13 reflected that established on Day 4.     

At 30 mg/kg bw/day, there was considered to be no effect on litter size and offspring survival from implantation to birth and subsequently to termination on Day 13 of age. However, although post-natal survival amongst litters maintained to termination was good, two females at this dosage did show total litter losspost partumearly in lactation.

At 15 mg/kg bw/day, the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age was unaffected by maternal treatment.


Offspring Growth and Development

At 60/90/60 mg/kg bw/day, mean body weight of the offspring on Day 1 and subsequent mean body weight gains to termination on Day 13 of age were slightly lower than control. Litter weights were lower than control throughout but principally reflected the lower litter sizes at this dosage compared to control.    

There was no effect of maternal treatment on offspring body weight on Day 1 or subsequent body weight gain to termination at 15 or 30 mg/kg bw/day. At both dosages, litter weights were lower than control but this was attributable to differences in litter sizes at these dosages compared to control.   

At 60/90/60 mg/kg bw/day and, to a lesser extent 30 mg/kg bw/day, the incidence of offspring clinical signs from birth to Day 4 of age was higher than control, although these clinical signs did not indicate any developmental effect on the offspring. At 60/90/60 mg/kg bw/day, offspring from four litters showed no indication of milk in the stomach; all offspring from three of these litters failed to survive to Day 4 and the other litter showed poor offspring survival.

The type and incidence of offspring clinical signs at 15 mg/kg bw/day did not indicate any effect of maternal treatment.

Ano-genital distance offspring on Day 1post partumand visible nipple count for male offspring on Day 13post partumwas unaffected by maternal treatment at 15, 30 or 60/90/60 mg/kg bw/day.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring did not indicate any obvious effect of maternal treatment on offspring development at 15, 30 or 60/90/60 mg/kg bw/day. 

At 60/90/60 mg/kg bw/day, a number of decedent offspring and offspring killed at Day 4 of age showed no milk in the stomach.    

Adults

Neither the type, incidence or distribution of necropsy finding for surviving animals indicated any obvious effect of treatment for either sex at 15 or 30 mg/kg bw/day, males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.

Organ Weights

For males at 60/90 mg/kg bw/day, absolute and body weight relative prostate weights were lower than control. 


Histopathology

Histopathological examination of reproductive tissues (testes, epididymides and ovaries) from the control animals, 60/90 mg/kg bw/day male animals and 60/90/60 mg/kg bw/day female animals did not reveal any findings considered to be related to treatment with the test item.

Thyroid Hormone Analysis

Levels of thyroxine (T4) in adult males did not indicate any effect of treatment or indication of endocrine disruption at 15, 30 or 60/90 mg/kg bw/day.

Levels of thyroxine (T4) in offspring at Day 13 of age did not indicate any effect of maternal treatment or indication of endocrine disruption at 15, 30 or 60/90/60 mg/kg bw/day.

Conclusion

Based on the results of this study, a dosage of 15 mg/kg bw/day Dichlorobis(n-cyclopentadienyl)titanium (CAS# 1271-19-8) is considered to be the No Observed Effect Level (NOEL) for adult toxicity, reproduction and the developing offspring. A dosage of 30 mg/kg bw/day may represent a No Observed Adverse Effect Level (NOAEL) for reproduction and the developing offspring.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One K1 study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

In an OECD 421 there were no direct adverse effects on development observed.

For the offspring, despite the higher offspring mortality and lower weight gain observed at 60/90/60 mg/kg bw/day, clinical signs and necropsy findings did not indicate any effect on morphological development. Ano-genital distance on Day 1 was unaffected and body weight gain of the offspring was only slightly lower than control. 

However high mortality of the offspring was observed during until Day 4 of age and while this was considered to reflect maternal toxicity rather than a selective effect on the offspring, the incidence of offspring mortality at this dosage was probably too high to considered this being a NOAEL for the offspring. At 30 mg/kg bw/day, the incidence of offspring mortality was lower, although two total litter losses did occur, and this dosage may represent a NOAEL for the offspring. The No Observed Effect Level (NOEL) for the offspring in this study was considered to be 15 mg/kg bw/day.

 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 21 December 2016 Experimental Completion Date: 14 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification : Dichlorobis(n-cyclopentadienyl)titanium
(CAS# 1271-19-8)
Physical State/Appearance : Red powder
Chemical Name : bis(cyclopentadienyl) titanium dichloride
Purity : 99.3%
Batch Number : 1504510829
Date Received : 21 June 2016
Storage Conditions : Ambient conditions, in the dark; used/formulated in light
Expiry Date : 03 April 2019
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury.
The animals were acclimatized for twenty six days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 271 to 347g and were approximately eleven weeks old and the females weighed 184 to 247g, and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Corn oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least eleven days when stored refrigerated (approximately 4°C) in the dark. Formulations were initially prepared on a daily basis until this stability was confirmed and then prepared on an approximately weekly basis and stored at approximately 4 °C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of Dichlorobis(n-cyclopentadienyl)titanium at Envigo Research Limited, Shardlow, UK, Analytical Services on four occasions during the study. The results indicate that the prepared formulations were within 99-108% of the nominal concentration confirming the suitability of the formulation procedure for the purpose of this study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item described in the main part of this study was also used as the analytical standard.

Preparation of Claibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot of test item was exactly weighed into a volumetric flask and brought to volume with dilution solvent. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL. Standard solutions contained the equivalent amount of vehicle to that of the relevant samples.

On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis sequence as a minimum.

To assess the calibration range of the method, a range of standard solutins were prepared in dilution solvent and vehicle covering the contration range 0.0496 mg/mL to 0.1848 mg/mL.

Preparation of Test Samples
The formulations received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent, which was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentrations.

Preparation of Accuracy and Precision Samples
Samples of Corn Oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis.

The concentration of Test Item in the final solution was quantified by HPLC using UV detection.

Instrumentation Parameters
HPLC: AgilentTechnologies 1200, incorporatng autosampler and workstation
Column: Luna 5 µ silica (250 x 4.6 mm id) at 30°C
Mobile phase: Tetrahydrofuran
Flow-rate: 1 mL/min
UV detector wavelength: 254 nm
Injection volume: 10 µL
Retention time: ~3.4 mins
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
approximately six weeks (males) and eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Daily
Duration of test:
approximately six weeks (males) and eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
high dosage being increased to 90 mg/kg bw/day from Day 10 and subsequently lowered back to 60 mg/kg bw/day for females only from Day 44.
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected in collaboration with the Sponsor Representative and were based on available toxicity data including preliminary data from a fourteen day dose range-finding toxicity study in the rat (Envigo Study Number YY06FT). In the range-finding study, dose levels of 30, 60 or 125 mg/kg bw/day resulted in lower overall body weight gains in a dose-related manner with the effect appearing to be more pronounced in males. This was associated with slightly lower overall food consumption in animals of either sex treated with 125 or 60 mg/kg bw/day with an increase in overall water consumption also being evident for the high dose males. At necropsy, macroscopic findings were confined to females from the 125 mg/kg bw/day dose group where 2/3 animals showed red discoloration of the glandular region in the stomach. A high dose level of 60 mg/kg bw/day was therefore initially considered to be suitable for investigation in this OECD 421 together with 15 and 30 mg/kg bw/day as the low and intermediate dose levels, respectively. Following a review of the available in-life data from the first week of dosing, the high dosage was increased to 90 mg/kg bw/day from 25 January 2017 (Day 10). Following the premature termination of two high dosage females and increased offspring mortality for a further high dosage litter, the high dosage for females was lowered to 60 mg/kg bw/day from 28 February 2017 (Day 44).
Maternal examinations:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period for the periods covering post partum Days 1-4, 4-7 and 7-14.

Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

Estrous Cycle Assessment
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Terminal Investigations
Necropsy
Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were killed via carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy were killed around the same time as littering females.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination; where external observations were detected an internal examination was performed.

Thyroid Hormone Assessment
Where possible, blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormone. Blood samples to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than
-60 ºC for possible evaluation of thyroid hormones. Samples were taken as follows:

Serum and plasma samples were taken from all adult males and all adult females at termination.

All serum samples were shipped to the test site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) for serum analysis, frozen, packed in dry ice. The serum from adult males and Day 13 offspring were analyzed for Thyroxine (T4) under the supervision of the Prinicpal Investigator. All plasma samples were retained at the Test Facility.

Organ Weights

Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed post-fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Cowpers glands
Pituitary
Epididymides ♦
Glans penis
Seminal vesicles (with coagulating gland)
Gross lesions
LABC (levator ani-bulbocavernous) muscle
Thyroid/parathyroid
Ovaries
Uterus/Cervix (and oviducts)
Mammary gland
Vagina

All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing. The tissues from control and high dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.
Ovaries and uterine content:
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Fetal examinations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
Statistics:
Please refer to "Any other information on materials and methods including tables"
Indices:
Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
Time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices
For each group the following were calculated:

Mating Index (%) = (No. of animals mated / No. of animals paired) x 100

Pregnancy Index (%) = (No. of pregnant females / No. of animals mated) x 100

Gestation and Parturition Data
Calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the no. of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:

Parturition Index (%) = (No. of females delivering live offspring / No. of pregnant females) x 100


Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).

i. Post-Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:

Post–implantation loss (%) = ((No. of implantation sites - Total no. of off-spring born) / No. of implantation sites) x100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Viability Index 1 (%) = (Nukber of offspring alive on Day 4 / Number of offpsring alive on Day 1) x 100

Viability Index 2 (%) = (Number of offspring alive on Day 7/13 / Number of offspring alive on Day 4) x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum:

(Number of male offpsring / Total number of offspring) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed for surviving animals that indicated any adverse effect of treatment for either sex at 15 or 30 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.
Increased post-dosing salivation was observed for the majority of males and, at a lower incidence, the majority of females at 60/90/60 mg/kg bw/day. This finding is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect unpalatability or slight irritancy of the test item formulation, rather than any systemic effect of treatment.

Noisy respiration was occasionally observed for isolated treated animals on sporadic occasions during the study. However, the distribution and incidence of this finding indicated that it was most probably associated with occasional difficulties in dosing animals rather than any indication of test item toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female number 89 was killed for animal welfare reasons on Day 41 of the study after showing piloerection, lethargy, hunched posture, dehydration and diarrhoea. Necropsy revealed red coloured contents in the stomach which appeared thin and distended and showed sloughing and raised white patches on the non-glandular region and red patches on the glandular region. Additionally, black contents were observed in the colon and cecum and the cecum also appeared small.

Female number 94 was killed for animal welfare reasons on Day 43 of the study after showing piloerection, lethargy, hunched posture, dehydration and staining of the fur and around the eyes and snout. Necropsy revealed pale kidneys and black colored contents in the stomach which showed sloughing, a raised limiting ridge and red patches on the glandular region. Additionally, black contents were observed in the cecum which appeared small.
Both of these deaths occurred around the time of parturition and the additional physiological stresses associated with delivery of a litter may have been a contributory factor in the decline in clinical condition of these animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment of females at dosages of 15, 30 or 60/90 mg/kg bw/day during the two week pairing phase was not associated with any effects on body weight or food consumption that were considered to be related to treatment. Statistically significant lower body weight gain was apparent at 30 and 90 mg/kg bw/day during Week 1, compared to control, but the extent of these differences considering the age of the animals and the lack of dosage relationship indicated that these findings were incidental and unrelated to treatment.

At 60/90 mg/kg bw/day, body weight gain and food consumption of females during gestation were statistically significantly lower than control. During late gestation, these values, particularly body weight gain, may been influenced by lower in utero litter size, however the lower female body weight observed at the start of lactation would tend to indicate an underlying effect of treatment on the females during late gestation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on food consumption of females during the pre-pairing phase of the study at 15, 30 or 60/90 mg/kg bw/day.

At 30 and 60/90 mg/kg bw/day, food consumption of females was statistically significantly lower than control throughout gestation, although the differences in food intake during late gestation may have been influenced, to a certain extent, by slightly lower litter sizes at these dosages compared to control. There was no effect on food consumption of females during gestation at 15 mg/kg bw/day.

At 60/90 mg/kg bw/day, lower food consumption, compared to control, was apparent for females throughout lactation, with the lower intakes attaining statistical significance during days 4-7 and 7-14 of lactation. However, the lower intake at this dosage was probably, at least in part, influenced by the lower litter size at this dosage compared to control.

At 15 and 30 mg/kg bw/day, food intake was also lower than control throughout lactation, however differences failed to attain statistical significance and may have also have been influenced by lower litter demand due to lower litter size at these dosage, compared to control.

Food consumption did attain statistical significance when compared with control on Days 4-7 and 7-14 of lactation, and these differences may well be attributable to ;pwer demand from the suckling litter.
Food efficiency:
no effects observed
Description (incidence and severity):
Intergroup differences in food conversion efficiency for females during the pre-pairing phase of the study did not indicate and effect of treatment at 15, 30 or 60/90 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate and effect of treatment.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences from control for thyroid weight of either sex at 15 or 30 mg/kg bw/day, for males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Neither the type, incidence or distribution of necropsy finding for surviving animals indicated any obvious effect of treatment for either sex at 15 or 30 mg/kg bw/day, for males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of reproductive tissues (testes, epididymides and ovaries) from the control and high dosage animals (60/90 mg/kg bw/day for males and 60/90/60 mg/kg bw/day for females) did not reveal any findings considered to be related to treatment with the test item. In particular, no consistent treatment-related pathologic findings in the testes following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or in the ovaries following the evaluation of the follicles and corpora lutea.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males did not identify any effect of treatment or indication of endocrine disruption at 15, 30 or 60/90 mg/kg bw/day.
Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 15, 30 or 60/90/60 mg/kg bw/day.
Number of abortions:
not examined
Description (incidence and severity):
Not applicable, as rats do not abort.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
At 60/90/60 mg/kg bw/day, the mean number of implantations was slightly lower than control but intergroup differences indicated that this finding was incidental and unrelated to treatment. Subsequently, the mean total number of offspring born was also slightly lower than control, however, while this finding is consistent with the previous lower implantation count, four females showed relatively high incidences of post-implantation losses. Three of these females (one of whom was later killed in extremis) subsequently showed total litter loss post partum. It should be noted that post-implantation loss may include offspring that were actually born but not observed before being cannibalized by the mother. Live litter size on Day 1 was clearly lower than control due to inferior offspring survival after parturition, as indicated by inferior mean live birth index compared to control. Subsequent offspring survival for litters surviving to termination was also inferior to control to Day 4 of age. These findings have to be considered in the context of four females (one of whom was subsequently killed in extremis) that showed total litter loss post partum during early lactation. Subsequent offspring survival from Day 4 to termination on Day 13 was similar to control and appeared unaffected by maternal treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
At 60/90/60 mg/kg bw/day, the distribution of gestation lengths attained statistical significance compared with control but, as this distribution was similar to that at 15 mg/kg bw/day, an association with treatment appears unlikely. One decedent female killed in extremis around the time of parturition did show a notably long gestation period (24 days) but it was unclear whether this extended gestation period was caused by the poor clinical condition of the female or was a contributing factor in the decline in the clinical condition of the animal. There was no obvious association between gestation length and the incidence of decedent females/females showing total litter loss post partum.
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 60/90/60 mg/kg bw/day, mean body weight of the offspring on Day 1 and subsequent mean body weight gain to termination on Day 13 of age was only slightly lower than control, with differences failing to attain statistical significance. Considering the smaller size of the litters, it may have been expected for offspring growth to show greater parity with their control counterparts. Litter weights were lower than control throughout lactation and, while this would have been influenced by lower offspring weight gain, it principally reflected the lower litter sizes at this dosage compared to control.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (actual dose received)
Treatment related:
no
Conclusions:
Based on the results of this study, a dosage of 15 mg/kg bw/day Dichlorobis(n-cyclopentadienyl)titanium (CAS# 1271-19-8) is considered to be the No Observed Effect Level (NOEL) for adult toxicity, reproduction and the developing offspring. A dosage of 30 mg/kg bw/day may represent a No Observed Adverse Effect Level (NOAEL) for reproduction and the developing offspring.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females). Dose levels of 15, 30 and 60 mg/kg bw/day were initially employed, with the high dosage being increased to 90 mg/kg bw/day from Day 10 and subsequently lowered back to 60 mg/kg bw/day for females only from Day 44. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil) over the same treatment period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with pregnant females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance on Day 1 and visible nipple count on Day 13 (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all females through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning of the day of termination for all females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues from control and high dose animals was performed.


Results…….

Adult Responses

Mortality

There were two unscheduled deaths on the study, both occurring for females at 60/90 mg/kg bw/day around the time of parturition. 

Female 89 was killed on Day 41 after showing piloerection, lethargy, hunched posture, dehydration and diarrhoea. Necropsy revealed red coloured contents in the stomach which appeared thin and distended and showed sloughing and raised white patches on the non-glandular region and red patches on the glandular region. Additionally, black contents were observed in the colon and cecum and the cecum also appeared small. 

Female 94 was killed on Day 43 of the study after showing piloerection, lethargy, hunched posture, dehydration and staining of the fur and around the eyes and snout. Necropsy revealed pale kidneys and black coloured contents in the stomach which showed sloughing, a raised limiting ridge and red patches on the glandular region. Additionally, black contents were observed in the cecum which appeared small.

Clinical Observations

There were no clinical signs observed for surviving animals that indicated any systemic effect of treatment for either sex at 15, 30 mg/kg bw/day or males at 60/90 mg/kg bw/day and females at 60/90/60 mg/kg bw/day.

Body Weight

At 60/90 mg/kg bw/day, body weight gains of males were generally lower than control from the second week of treatment, resulting in lower body weight and overall body weight gain at the end of the study.

There was no effect of treatment on body weight gain of males at 15 or 30 mg/kg bw/day throughout the study. 

There was no effect of treatment on body weight gain of females during the pre-pairing phase of the study at 15, 30 or 60/90 mg/kg bw/day. 

At 60/90 mg/kg bw/day, body weight gain of females during gestation was lower than control, although the differences from control observed during late gestation were, in part, attributable to a lower contribution from the gravid uterus due to slightly lower litter size at this dosage compared to control.  

At 30 mg/kg bw/day, body weight gain of females during the last week of gestation was slightly lower than control, resulting in lower overall body weight gain at the end of gestation. These differences from control were probably attributable to lower contribution from the gravid uterus due to the slightly lower litter size at this dosage compared to control.   

At 15 mg/kg bw/day, overall body weight gain of females during gestation was lower than control but, was influenced by a lower contribution from the gravid uterus due to the slightly lower litter size at this dosage, compared to control.  

At 60/90/60 mg/kg bw/day, body weight gain of females during lactation was lower than control, despite a probable lower demand on the females from the smaller litters at this dosage compared to their control.

At 15 and 30 mg/kg bw/day, body weight gain during lactation was unaffected by treatment.  

Food Consumption

There was no effect of treatment on food consumption of males throughout the study at 15, 30 or 60/90 mg/kg bw/day.

There was no effect of treatment on food consumption of females during the pre-pairing phase of the study at 15, 30 or 60/90 mg/kg bw/day.

At 30 and 60/90 mg/kg bw/day, food consumption of females was lower than control throughout gestation but, to some extent, may have been influenced by slightly lower litter sizes at these dosages compared to control. At 15 mg/kg bw/day, there was no effect of treatment on food consumption of females during gestation.

At 60/90/60 mg/kg bw/day, food consumption of females was lower than control throughout lactation, but, to some extent, may have been influenced by slightly lower litter sizes at this dosage compared to control.

At 15 and 30 mg/kg bw/day, food intake was slightly lower than control throughout lactation, but differences were probably attributable to lower litter demand due to lower litter size at these dosages, compared to control.  

Food Conversion Efficiency

At 60/90 mg/kg bw/day, food conversion efficiency for males appeared inferior to control during the second week of treatment (pre-pairing) and during the post-pairing phase of the study. At 15 and 30 mg/kg bw/day, food conversion efficiency for males appeared unaffected by treatment.

Food conversion efficiency for females during the pre-pairing phase of the study was unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day. 

Water Consumption

Visual inspection of water bottles did not indicate any effect of treatment for either sex throughout the study.      


Reproductive Performance

Estrous Cycle

Estrous cycles during the pre-pairing phase of the study were unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Mating

Mating performance pre-coital interval was unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Fertility

Fertility, as assessed by the number of females that achieved pregnancy, was unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Gestation Length

Gestation length appeared unaffected by treatment at 15, 30 or 60/90 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

At 60/90/60 mg/kg bw/day, the mean number of offspring born was slightly lower than control but consistent with previous implantations count. Four females showed relatively high incidences of post-implantation losses and three of these females (including one female which was later killedin extremis) subsequently showed total litter losspost partum. Mean live litter size on Day 1 was lower than control due to inferior offspring survival, indicated by lower mean live birth index. Subsequent offspring survival was also inferior to control to Day 4 of age, indicated by lower Day 4 viability index. The lower live litter size on Days 1 and 4 have to be considered in the context of four other females (one of whom was subsequently killedin extremis) that showed total litter losspost partumshortly after parturition. Subsequent offspring survival from Day 4 for surviving litters was unaffected by maternal treatment, therefore the lower litter size on Day 7 and 13 reflected that established on Day 4.     

At 30 mg/kg bw/day, there was considered to be no effect on litter size and offspring survival from implantation to birth and subsequently to termination on Day 13 of age. However, although post-natal survival amongst litters maintained to termination was good, two females at this dosage did show total litter losspost partumearly in lactation.

At 15 mg/kg bw/day, the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age was unaffected by maternal treatment.


Offspring Growth and Development

At 60/90/60 mg/kg bw/day, mean body weight of the offspring on Day 1 and subsequent mean body weight gains to termination on Day 13 of age were slightly lower than control. Litter weights were lower than control throughout but principally reflected the lower litter sizes at this dosage compared to control.    

There was no effect of maternal treatment on offspring body weight on Day 1 or subsequent body weight gain to termination at 15 or 30 mg/kg bw/day. At both dosages, litter weights were lower than control but this was attributable to differences in litter sizes at these dosages compared to control.   

At 60/90/60 mg/kg bw/day and, to a lesser extent 30 mg/kg bw/day, the incidence of offspring clinical signs from birth to Day 4 of age was higher than control, although these clinical signs did not indicate any developmental effect on the offspring. At 60/90/60 mg/kg bw/day, offspring from four litters showed no indication of milk in the stomach; all offspring from three of these litters failed to survive to Day 4 and the other litter showed poor offspring survival.

The type and incidence of offspring clinical signs at 15 mg/kg bw/day did not indicate any effect of maternal treatment.

Ano-genital distance offspring on Day 1post partumand visible nipple count for male offspring on Day 13post partumwas unaffected by maternal treatment at 15, 30 or 60/90/60 mg/kg bw/day.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring did not indicate any obvious effect of maternal treatment on offspring development at 15, 30 or 60/90/60 mg/kg bw/day. 

At 60/90/60 mg/kg bw/day, a number of decedent offspring and offspring killed at Day 4 of age showed no milk in the stomach.    

Adults

Neither the type, incidence or distribution of necropsy finding for surviving animals indicated any obvious effect of treatment for either sex at 15 or 30 mg/kg bw/day, males at 60/90 mg/kg bw/day or females at 60/90/60 mg/kg bw/day.

Organ Weights

For males at 60/90 mg/kg bw/day, absolute and body weight relative prostate weights were lower than control. 


Histopathology

Histopathological examination of reproductive tissues (testes, epididymides and ovaries) from the control animals, 60/90 mg/kg bw/day male animals and 60/90/60 mg/kg bw/day female animals did not reveal any findings considered to be related to treatment with the test item.

Thyroid Hormone Analysis

Levels of thyroxine (T4) in adult males did not indicate any effect of treatment or indication of endocrine disruption at 15, 30 or 60/90 mg/kg bw/day.

Levels of thyroxine (T4) in offspring at Day 13 of age did not indicate any effect of maternal treatment or indication of endocrine disruption at 15, 30 or 60/90/60 mg/kg bw/day.

Conclusion

Based on the results of this study, a dosage of 15 mg/kg bw/day Dichlorobis(n-cyclopentadienyl)titanium (CAS# 1271-19-8) is considered to be the No Observed Effect Level (NOEL) for adult toxicity, reproduction and the developing offspring. A dosage of 30 mg/kg bw/day may represent a No Observed Adverse Effect Level (NOAEL) for reproduction and the developing offspring.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One K1 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

Based on the available information the substance is not classified for reproductive toxicity.