Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 2017 until July 2018 (unaudited draft report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Albemarle Germany GmbH, batch No. 78562
- Expiration date of the lot/batch: 31.12.2020
- Purity test date: 94% Titanium, H min 3.7%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature under water and/or inert atmosphere (N2 or Ar)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: suspension, stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: only with air

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl San Pietro al Natisone UD, Italy order supply from Envigo Netherlands Kreuzelweg 53 5961 NM Horst, NL
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 to 8 weeks
- Weight at study initiation: (P) Males: 212 - 223 g; Females: 196 -205 g
- Fasting period before study: no
- Housing:
The animalswere housed in a limited access rodent facility.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone
solid bottomed cages measuring 59.5×38×20 cm. Nesting material was provided inside suitable bedding bags and changed at least
twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages
measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected
and changed daily.
After mating, the males were re-caged as they were before mating.
The femaleswere transferred to individual solid bottomed cages (measuring 42.5×26.6×18.5
cm) for the gestation period, birth and lactation. Nesting material was provided inside
suitable bedding bags.
Recovery animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed
cages measuring 59.5×38×20 cm. Nesting
material was provided inside suitable bedding bags and changed at least twice a week.
- Diet ad libitum
- Water ad libitum
- Acclimation period: 29 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 h
IN-LIFE DATES: From Oct. 26, 2017 to February 8, 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% Carboxymethylcellulose in water to maintain a stabil suspension of the unsoluble test substance
- Concentration in vehicle: 10, 30, 100 mg/mL.
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Vaginal smears were taken from the
day after the start of pairing until positive identification of copulation (spermidentification,
vaginal plug in situ or copulation plug found in the cage tray).
- Proof of pregnancy: [vaginal plug or copulation plug in the cage] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how):
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Remarks:
dififculties in establishing and reproducing the analytical results and recovery were encountered.
Details on analytical verification of doses or concentrations:
A detailed analytical report was provided but doses were established based on nominal concnetrations in the dosing solutions that were prepared freshly every day.
Duration of treatment / exposure:
Males of the main groups were treated for 2 weeks prior to pairing and during pairing with
females until the day before necropsy, for a total of 29 days.
Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum
and post partum periods until Day 13 post partum (for at least 51 days). Two not pregnant
females were dosed up to the day before necropsy.
Frequency of treatment:
Once daily
Details on study schedule:
Main groups
Males of the main groups were treated for 2 weeks prior to pairing and during pairing with
females until the day before necropsy, for a total of 29 days.
Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum
and post partum periods until Day 13 post partum (for at least 51 days). Two not pregnant
females were dosed up to the day before necropsy.
The following investigations were performed: mortality check, clinical signs (including
neurotoxicity assessment, motor activity and sensory reactivity to stimuli), body weight,
food consumption, oestrous cycle, mating performance, clinical pathology investigations
(haematology comprising coagulation, clinical chemistry and also urinalysis in males only),
litter data, sex ratios, macroscopic observations and organ weights. Histopathological examination was performed in control and high dose groups (five animals/sex/group
randomly selected), aswell as on all abnormalities detected during post mortem observation
at the end of treatment period. The identification of the stages of the spermatogenic cycle
was also performed in five randomly selected males of the control and high dose groups.
Thyroid hormone determination was performed in all males.
Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination
of 1/pup/sex/litter (where possible) at Day 14 post partum were determined.
All pups found dead in the cage were examined for external and internal abnormalities. All
culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex
was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were examined for external abnormalities
and sex confirmation by gonadal inspection.
Recovery groups
Recovery animals (males) were treated for up to 4 consecutive weeks and killed after 6
weeks of recovery period.
Recovery animals (females) were treated for up to 6 weeks, when most of the main group
females were killed. The females were sacrificed after 6 weeks of recovery period.
The following parameters were evaluated in these animals: mortality check, clinical signs
(including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to
stimuli), body weight, food consumption, macroscopic observations and organ weights.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
control group
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose group
Dose / conc.:
0 mg/kg bw/day
Remarks:
recovery control
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Recovery group
No. of animals per sex per dose:
10 males, 10 females in the main groups, 5 males 5 feamles in the recovery groups.
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were based on a 14 day dose range finding study.
Positive control:
no tapplicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, mornign and afternoon for moratlity, clinical signs once before treatment and atleast once daily.
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: FOB Once before commencement of treatment and a least once per week.
Each animal was removed from the home
cage and observed in an open arena. The tests included observation of changes in gait and
posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre
behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection,
pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.
Grip strength and sensory reactivity to stimuli (All groups)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly
selected from each group for evaluation of sensory reactivity to stimuli of different
modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip
strength.
For the females the tests were performed on Day 12 post partum (where possible).
Measurements were performed using a computer generated random order (for the main
groups).
Once during Week 6 of recovery, these evaluations will also be performed in all animals.
Motor activity assessment (MA) - (All groups)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly
selected from each group and the motor activity was measured (for approximately
5 minutes) by an automated activity recording device.
For the females the tests were performed on Day 12 post partum (where possible).
Measurements were performed using a computer generated random order (for the main
groups).
Once during Week 6 of recovery, the MA was also performed in all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Main groups: Males weighed weekly from allocation to termination, Females weighed weekly from allocation to positive identification of mating and on
Days 0, 7, 14 and 20 post coitum. Dams also weighed on Days 1, 4, 7, 13 post
partum and just prior to necropsy.
Recovery groups: Each animal weighed on the day of allocation to treatment groups, on the day that
treatment commences, weekly thereafter and just prior to necropsy.
FOOD CONSUMPTION :
Main groups: The weight of food consumed by each cage of males and females was recorded weekly
(whenever possible) during the pre-mating period starting from allocation. Individual food
consumption for the females was measured on Days 7, 14 and 20 post coitum starting
from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.
Recovery groups: The weight of food consumed by each cage of rats will be recorded at weekly intervals
following allocation.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):. No
Estrous cyclicity (parental animals):
Stock females: .Oestrus cycle were monitored by vaginal smears in all stock females for at least 1 week
before allocation in order to exclude from the study females with irregular cycle.
Females in the test groups: Vaginal smears will be taken in the morning from Day 1 of treatment, up to positive
identification of mating including no less than 2 weeks before the pairing.
Females allocated to groups
The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females, before dispatch to necropsy. No vaginal
smears were foreseen to be taken from females sacrificed for humane reasons.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, seminal vesicles weight, prostate weight. Histopathology Epididymes, penis, seminal vesicles, testes, coagulating gland, prostate. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled
to 8 (4 males and 4 females, where possible) by a random selection. At least two pups
(males or females, selected for culling) were sacrificed for blood collection.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized tothe cube root of body weight collected on Day 1 post partum. Nipple count on day 13 post partum.

All culled pups sacrificed at Day 4 post partum were subjected to an external examination.Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalitiesand sex confirmation by gonadal inspection.
Thyroid: Pups at Day 14 post partum
Thyroid was weighedat day 14 post partum from one male and one female pups selected for blood collectionof hormone determination and preserved in 10% neutral buffered formalin. The thyroid
weights were determined after fixation.
GROSS EXAMINATION OF DEAD PUPS:
yes, All pups found dead in the cage were examined for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals:The males were killed after the mating of all females on Day 30 of the study.
- Maternal animals: The females with live pups were killed on Day 14 post partum. The non-pregnant females
were killed on Day 28 post coitum
Recovery animals: Animals were killed after 6 weeks of recovery.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring: Pups that had completed the scheduled test period (Day 4 or 14 post partum) were euthanised
by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection
for hormone determinationwere killed on the day of blood sampling.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities
and sex confirmation by gonadal inspection.
GROSS NECROPSY
- All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities
and sex confirmation by gonadal inspection.
HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid was weighed from one male and one female pups selected for blood collection
of hormone determination and preserved in 10% neutral buffered formalin. The thyroid
weights were determined after fixation.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance
of the differences amongst group means was assessed by Dunnett’s test or a modified
t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis
of variance (non-continuous variables) was used for the other parameters. Intergroup
differences between the control and treated groups were assessed by the non-parametric
version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean
values, standard deviations and statistical analysis were calculated from actual values in
the computer without rounding off.
Reproductive indices:
Males
Males:
C opul a t i on I nd e x (%) = no.of animals mated /no.of males paired ×100
F e r t i l i t y I nd e x (%) = no.of males which induced pregnancy / no.of males paired x 100

Females
C opul a t o r y I nd e x (%) = no.of animals mated / no.of females paired ×100
F e r t i l i t y I nd e x (%) = no.of pregnant females / no.of females paired ×100
Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:
no. of corpora lutea − no.of implantations / no. o f corpora lutea ×100
Pre-natal loss on Day 0 post partum was calculated as a percentage from the formula:
no.of visible implantations − live litter size at birth / no.of visible implantations x 100
Post-natal loss on Day 4 post partum, before culling, was calculated as a percentage from
the formula:
Live litter size at birth − live litter size at Day 4, before cull ng /Total litter size at birth x 100
Post-natal loss on Day 13 post partum, after culling, was calculated as a percentage from
the formula:
Live litter size on Day 4 (after cul l ing) − live littersize on Day 13/ Live litter size on Day 4 (after culling ) x100
Sex ratios were calculated at birth, on Day 4 and on 14 post partum and are presented as
the percentage of males per litter.
Anogenital distance was calculated on Day 1 post partum and normalised to the cube root
of pup weight performed on Day 1 post partum.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Main groups
No clinical signs were observed throughout the study in main group animals of both sexes.
Recovery groups
During treatment or during the recovery phase, no significant clinical signs were observed. Damaged hindlimb observed in one control male (no. X0700090, Group 5) from the last day
of treatment up to the end of the recovery period and damaged eye observed in one control female (no. X0700089, Group 5) on the last day of the recovery period were considered
incidental findings.
FOB:
Main and Recovery groups
Neurotoxicity assessment (removal of animals from the home cage and observations in open arena) did not reveal changes attributable to the test item in treated animals of both
sexes, when compared to the control group.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Main groups
No significant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.
Recovery groups
No relevant differences in body weight and body weight gain were noted between control and the treated groups both in males and females throughout the treatment and recovery
phase.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Main groups
No significant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.
Recovery groups
No relevant differences in body weight and body weight gain were noted between control and the treated groups both in males and females throughout the treatment and recovery phase.
Food efficiency:
no effects observed
Description (incidence and severity):
Main and Recovery groups
Food consumption was unaffected by treatment in both sexes during the study.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing phase -Main groups
Leucopenia was recorded in some males dosed at 300 and 1000 mg/kg/day. Mean group values were 28% and 34% below controls, respectively, showing some dose-relation.
The statistically significant differences between control and treated females (mean corpuscular volume, mean corpuscular haemoglobin and leucocytes) were of minimal severity
and/or not dose-related, therefore they were considered to be of no toxicological relevance.
Recovery phase - Recovery groups
Changes recorded during the dosing phase were no longer observed. The statistically significant decrease of platelets observed in males dosed at 1000 mg/kg/day
was not recorded at the dosing phase, therefore it was considered unrelated to treatment.

Coagulation
Dosing phase -Main group
Statistically significant differences of activated partial thromboplastin timewere recorded in males dosed at 300 mg/kg/day and females receiving 100 mg/kg/day, compared to controls.
Changes were not dose-related, therefore they were considered incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Dosing phase -Main groups
Compared with controls, males dosed at 1000 mg/kg/day showed decrease of alanine aminotransferase (17%) and phosphorus (9%), and increase of triglycerides (56%). Phosphorus
was also decreased in males receiving 300 mg/kg/day (8%). The severity of the findings observed was not considered to be suggestive of tissue/organ injury.

Recovery phase - Recovery groups
No differences between control and treated animals were recorded, confirming reversibility.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Dosing phase -Main groups (males only)
No changes were observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Main and Recovery groups
Neurotoxicity assessment (removal of animals from the home cage and observations in open arena) did not reveal changes attributable to the test item in treated animals of both
sexes, when compared to the control group.
Motor activity, grip strength and sensory reaction to stimuli:
Main and Recovery groups
No significant alterations in motor activity, grip strength or sensory reaction to stimuli were observed in any treatment group at the examinations performed during the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Final and recovery sacrifice
No relevant changes were observed on terminal body, absolute and relative organ weight of treated animals that completed the treatment or recovery period, when compared to the controls
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Final and recovery sacrifice
No remarkable differences were noted at post mortem examination in treated animals,when compared with controls, either in animals that completed the treatment or recovery
period.
Gross lesions such as reduced size of thymus or single, dark depressed areas in the glandular region of the stomach or enlarged ovaries, mainly noted in control and high dose females
that completed the treatment period (final sacrifice), were considered to be related to a physiological variation in pregnancy and/or stress related often noted in this kind of studies.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Final sacrifice
No treatment-related changes were noted in animals sacrificed at the end of the treatment period.
The sporadic lesions, reported in control and treated animals, were considered to be an expression of spontaneous/or incidental pathology or physiological changes,seen in this
species and age in this kind of studies and in our experimental conditions.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone determination:
Parental males
No changes were observed.
Pups on day 14 post partum:

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Oestrous cycle, pre-coital intervals and copulatory index were similar between treated and control animals.

Compared with controls, triiodothyronine was increased in some female pups of all treated groups. The increases were 1.9 to 3 fold, with no dose-relation.
Since no related changes of the other hormones were recorded, the above mentioned finding was considered to be incidental.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
The male copulatory index was 100% in all gropus.
Fertility index both for males and females was 100% each in Groups 1, 2 and 4 and 80% in Group 3. Two mid-dose males (nos. X0700044 and X0700048) failed to induce pregnancy.
These cases were considered incidental.
Implantation, pre-birth loss data and gestation length of females:
Gestation periods were similar between treated and control females. All pregnant dams
gave birth on Day 22 post coitum (mean value).
Corpora lutea, implantations, live litter size and pre-natal loss (percentage) were similar in control and treated groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
haematology
clinical biochemistry
Remarks on result:
other: Very slight changes in some hematology and clincal chemistry parameters in the high dose group that were reversible during the recovery period are not regarded as adverse effects.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related effects. Pre-weaning clinical signs were comparable between treated and control groups.
Apparently no food intake (milk), cold to touch, small and pale appearance were the main clinical signs noted in control and treated pups. Found dead or missing pups were also
observed both in control and treated groups. Black staining on the head or muzzle were also noted in two pups belonging to control or low dose groups.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Gestation periods were similar between treated and control females. All pregnant dams
gave birth on Day 22 post coitum (mean value). Corpora lutea, implantations, live litter size and pre-natal loss (percentage) were similar incontrol and treated groups.

Litter data at birth, on Day 1 and Day 4 post partum (before culling) and on Day 13 (after culling) post partum and sex ratios:
Litter data and sex ratios were unaffected by treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Litter and mean pup weights on Day 1 an d 4 post partum were comparble between dose and control groups.
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:
no effects observed
Description (incidence and severity):
No treatment-related differences were noted in thyroid hormones levels in pups at Day 14 post partum.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No negative effects were seen between the control and treated pups in anogenital distance. No treatment-related changes were seen between in thyroid weight of control and treated pups. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were seen between in thyroid weight of control and treated pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.
Histopathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be
1000mg/kg/day for both males and females. No adverse effects were observed up to the highest recommended dose level tested of 1000 mg/kg bw/day.
Executive summary:

The toxic effects on Sprague Dawley rats after repeated dosing with Titanium Hydride Powder VM, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated. in a study according to OECG TG 422 and GLP. A 6 week treatment free period was allowed in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase.

The vehicle was 1% aqueous solution of carboxymethylcellulose (10 mL/kg).

The study design was as follows: In the main group, groups of 10 males and 10 female per group were administered the test substance at doses of 0 (vehicle control) 100, 300 and 1000 mg/kg bw/day once daily by oral gavage. The recovery group animals (5 per sex per group) were adimistered 0 (vehicle control) and 1000 mg/kg bw/day once daily by oral gavage.

Main groups

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29 days.

Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). Two not pregnant females were dosed up to the day before necropsy.

The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), body weight,

food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology comprising coagulation, clinical chemistry and also urinalysis in males only),

litter data, sex ratios, macroscopic observations and organ weights. Histopathological examination was performed in control and high dose groups (five animals/sex/group

randomly selected), as well as on all abnormalities detected during post mortem observation at the end of treatment period. The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups.

Thyroid hormone determination was performed in all males.

Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of 1/pup/sex/litter (where possible) at Day 14 post partum were determined.

All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.

All live pups sacrificed on Day 14 post partum were examined for external abnormalities and sex confirmation by gonadal inspection.

Recovery groups

Recovery animals (males) were treated for up to 4 consecutive weeks and killed after 6 weeks of recovery period. Recovery animals (females) were treated for up to 6 weeks, when most of the main group females were killed. The females were sacrificed after 6 weeks of recovery period.

The following parameters were evaluated in these animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to

stimuli), body weight, food consumption, macroscopic observations and organ weights.

Results:

Parental animals

Mortality : No martality occured throughout the study.

Clinical signa, neurotoxicity including motor activity, grip strength and sensory activity to stimuli:

Main and recovery groups

No clinical signs were observed throughout the study in all main phase animals of both

sexes.

Neurotoxicity assessment :(removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups.

Recovery animals did not show significant clinical signs during treatment, as well as during the recovery phase. Observation of treated animals at removal from the cage and in an open arena (neurotoxicity assessment), as well as, in motor activity, grip strength and sensory reactivity to stimuli did not show differences between control and treated groups.

Body weights and body weight gains:

No significant differences in body weights and body weight gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and

recovery groups.

Food consumption:

No effects on food consumption were observed in either males or females throughout the

study, both for main and recovery groups.

Hematology:

Leucopenia recorded in some main group males dosed at 300 and 1000 mg/kg/day was no longer observed at the end of treatment confirming complete reversibility.

No treatment-related changes were recorded in coagulation parameters evaluated.

Clinical chemistry

Changes observed in high dose main group males (decrease in alanine aminotransferase and increase of triglycerides) and increase in phosphorus in mid- and high dose main group

males were no more seen at the end of the recovery period. In addition, the severity of the changes observed was not considered to be suggestive of tissue/organ injury.

Urinalysis:

No changes were observed in urine analysis performed in male animals.

Thyroid hormones

No treatment-related differences were noted in hormones levels in parental males and in pups at Day 14 post partum.

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show anydifferences that could be related to treatment.

Implantation, pre-birth loss data and gestation length of females

Gestation periods were similar between treated and control females. All pregnant dams gave birth on Day 22 post coitum (mean value). Corpora lutea, implantations, live litter size and pre-natal loss (percentage) were similar in control and treated groups.

Pup/Litter data:

Litter data and sex ratios were unaffected by treatment.

There were no compound-related effects. Pre-weaning clinical signs were comparable between treated and control groups.

No negative effects were seen between the control and treated pups in anogenital distance.

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

No treatment-related changes were seen between in thyroid weight of control and treated pups.

No treatment-related differences were seen in thyroid hormone levels between control and treated pups on day 14 paost partum.

No nipples were observed in male pups on Day 14 post partum.

In conlusion no adverse effect on parental animals and offspring were observed up to the highest dose of 1000 mg/kg bw of titanium hydride teste din this study. This is the maximum recommened dose level for repeated dose toxicity studies.