Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
14-days oral dose range finding study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 2017 to November 2017 (2weeks oral study) - Main test still on going until July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018
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)
Version / remarks:
adopted on 29 July 2016
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
* 2 weeks preliminary oral toxicity study in rats
- Identity: Titanium Hydride Powder VM
- Batch no.: 78562
- Expiry date: February 2019
- Appearance: Grey-ash/Black powder
- Storage conditions: Room temperature
- RTC number: 15399
* Main test
**STILL ON GOING - DRAFT REPORT EXPECTED END JULY 2018**

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat is the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals and environmental conditions:
Procedures and facilities will comply with the requirements of the Directive 2010/63/EU on the protection of animals used for scientific purposes. The national transposition of the Directive is defined in Decreto Legislativo 26/2014.
RTC test facility is fully accredited by AAALAC. Aspects of the protocol concerning animal welfare have been approved by RTC animal-welfare body.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item is administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals will receive the vehicle alone at the same dose volume.
The dose will be administered to each animal on the basis of the most recently recorded body weight and the volume administered will be recorded for each animal.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on oral exposure:
The required amount of Titanium Hydride Powder VM will be suspended in the vehicle. The formulations will be prepared daily. Concentrations will be calculated and expressed in terms of test item as supplied. *** TO BE COMPLETED ONCE MAIn TEST FINALIZED - JULY 2018***
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Not available yet ** Draft report available in July 2018**
Duration of treatment / exposure:
* Males
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter at least until the minimum total dosing period of 28 days has been completed including the day before necropsy. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.

* Females
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes will be calculated according to the last recorded body weight.

* Recovery groups (Groups 5 and 6)
Male animals will be dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. Female animals will be dosed once a day, 7 days a week, up to the scheduled kill of the majority of litters (maximum of approximately 9 weeks of treatment). Dose volumes will be adjusted once per week for each animal according to the last recorded body weight. No treatment will be given during the recovery period.
Frequency of treatment:
* Males: Animals will be dosed once a day.
* Females: Animals will be dosed once a day.
* Recovery groups: Male animals will be dosed once a day.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS (Study E0145).
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS (Study E0145).
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS (Study E0145).
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
MAIN STUDY (Study X0700)
No. of animals per sex per dose:
- Four groups (Group 1, Group 2, Group 3 and Group 4) containing: 10 male and 10 female rats
- Groups 5 and 6: Control (Group 5) and high dose level (Group 6) will include 5 additional animals per sex to be sacrificed after 4 weeks of recovery.
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
***TO REVISED AT STUDY COMPLETION - MADE BASED ON THE PROTOCOL OF X0700***

* Mortality (All groups)
Throughout the study, all animals will be checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure will be followed except that the final check will be carried out at approximately mid-day. This will allow post mortem examinations to be carried out during the working period of that day. Severely debilitated animals will be observed carefully. Animals judged to be in extremis will be killed. A complete necropsy will be performed in all cases as detailed below.

* Clinical signs (All groups)
Once before commencement of treatment and at least once daily during the study, each animal will be observed and any clinical signs will be recorded. Observations will be performed at the same time interval each day, the interval will be selected taking into consideration the presence of post-dose reactions.

* Clinical observations (Functional Observation Battery Tests) - (All groups)
Once before commencement of treatment and at least once a week thereafter, each animal will be given a detailed clinical examination. Each animal will be removed from the home cage and observed in an open arena. The tests will include 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 will be 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 will be 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 will be performed on Day 13 post partum (where possible). Measurements will be 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 will be randomly selected from each group and the motor activity will be measured (for approximately 5 minutes) by an automated activity recording device. For the females the tests will be performed on Day 13 post partum (where possible). Measurements will be performed using a computer generated random order (for the main groups). Once during Week 6 of recovery, the MA will also be performed in all animals.

* Body weight (All groups)
- Main groups
Males will be weighed weekly from allocation to termination. Females will be weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams will also be weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
- Recovery groups
Each animal will be weighed on the day of allocation to treatment groups, on the day that treatment commences, weekly thereafter and just prior to necropsy.

* Food consumption (All groups)
The weight of food consumed by each cage of males and females will be recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females will be 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.

* Clinical pathology investigations
As a part of the sacrificial procedure, samples of blood will be withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters, if possible) randomly selected from each main group, under condition of food deprivation. At the end of Week 6 of the recovery period, blood samples may also be taken (after consultation with the Sponsor) from all surviving animals under identical conditions in order to re-evaluate any parameters which show treatment-related changes at measurements performed during the treatment period. In addition, as part of the necropsy procedure, blood samples will be taken from the abdominal vena cava of all parental male and female rats from each main group and from animals of the recovery groups, under isofluorane anaesthesia, to perform the determination of serum T3, T4 and TSH levels.

The blood samples collected will be divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements to be performed on blood samples are listed below:

* Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count - Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

These parameters will be analysed by Siemens Advia 120.

* Coagulation tests
Prothrombin time
Activated partial thromboplastin time

These parameters will be analysed by Instrumentation Laboratory ACL 3000 PLUS.

* Clinical chemistry
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Inorganic phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

These parameters will be analysed by Siemens Advia 1200.

* Urinalysis (5 males randomly selected)

Individual overnight urine samples will also be collected during the last week of treatment from the same male animals selected for the clinical pathology investigations and under the same conditions. Before starting urine collection water bottles will be removed from each cage and each animal will receive approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

These parameters will be analysed by Menarini AUTION MAX 4280/AUTION ELEVEN AE 4020.
Sacrifice and pathology:
***TO REVISED AT STUDY COMPLETION - MADE BASED ON THE PROTOCOL OF X0700***
Parental animals in extremis, killed for humane reasons and those that have completed the scheduled test period and selected for blood collection, will be killed by exsanguination under isofluorane anaesthesia.

- Parental males (Main groups)
The males will be killed after the mating of all females or after at least 28 days of treatment period.

- Parental females (Main groups)
The females with live pups will be killed on Day 14 post partum.
The females with total litter loss will be killed on the day of the occurrence of total litter loss or shortly after.
The females showing no evidence of copulation will be killed after 25 days of the last day of the mating session.
The females which do not give birth 25 days after positive identification of mating will be killed shortly after.

- Males and females (Recovery groups)
Animals will be killed after 6 weeks of recovery.
Statistics:
***TO REVISED AT STUDY COMPLETION - MADE BASED ON THE PROTOCOL OF X0700***
Standard deviations will be calculated as appropriate. For continuous variables the significance of the differences amongst group means will be assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings will be carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5. The non-parametric Kruskal-Wallis analysis of variance will be used for the other parameters. Intergroup differences between the control and treated groups will be assessed by the non-parametric version of the Williams test. The criterion for statistical significance will be p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Mortality:
no mortality observed
Description (incidence):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Body weight and weight changes:
no effects observed
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Haematological findings:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Clinical biochemistry findings:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Urinalysis findings:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Immunological findings:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Gross pathological findings:
no effects observed
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Neuropathological findings:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
***RESULT of the 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
Final draft report available on July 2018
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Remarks on result:
other: Draft report of the study available July 2018

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
*** CONCLUSION ON 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS***
The toxicity of Titanium Hydride Powder VM was investigated in Sprague Dawley rats after daily oral administration for 2 weeks, at dose levels of 100, 300 and 1000 mg/kg/day. An additional group of animals was treated with the vehicle (1% aqueous solution of carboxymethylcellulose) and acted as a control. No mortality occurred and no clinical signs were observed in all treated animals; body weight and food consumption, as well as terminal body weight and organ weights were not affected by treatment. No treatment-related findings were detected at post mortem examination (terminal body weight, organ weights, macroscopic and microscopic examinations) in treated animals, when compared with controls. Based on the results obtained in this study, the high dose level of 1000mg/kg/day could be selected as the high dose level for the subsequent reproductive toxicity study in rats.