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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-21 to 2014-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines, which do not affect the quality of the relevant results.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Chemical Name: Yttrium Oxide
Physical state: Fine Powder (at room temperature)
Color: white

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Details on animals:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of
the treatment period: males: 10-11 weeks old. females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups: males: 250 - 298 g (mean: 272.80 g, ± 20% = 218.24 – 327.36 g) - females: 162 - 199 g (mean: 183.13 g, ± 20% = 146.50 – 219.75 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF).

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 3°C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in Individually Ventiled cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %
Details on oral exposure:
Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Experimental Groups and Doses
According to the results of the dose range finding studyin which no overt toxicological changes were observed at 1000 mg/ kg body weight/ day, the following doses: 100 ; 300 and 1000 mg/kg bw were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Formulation Analysis
Each dosing concentration was analyzed for nominal concentration by ICP - optical emission spectroscopy using a validated analytical procedure. Stability and homogeneity of the test item in the vehicle was analyzed for the LD, MD and HD dosing formulation.
Samples for the nominal concentration verification was taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) of control and all treatment groups.
Samples for homogeneity were taken from the top, middle and bottom of HD, MD, and LD preparation in study week 1 and 5.
All formulation samples were stored at -20 oC until the analysis.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period (approximately 21 to 23 days) and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days (5 males from each group received dose administration for 28 days and the rest 5 animals from each group received dose administration for 29 days) was completed.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same dose volume
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
According to the results of the dose range finding study and in consultation with the sponsor three selected doses were tested for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

Examinations

Observations and examinations performed and frequency:
Body Weight and Food Consumption
The body weight of all animals were recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males. The food consumption in males were measured only for two week during premating period and not during the postmating period as the number of days during the postmating period are not uniform in all males due to difference in mating.

Mating
Mating was performed in a ratio of 1:1 (male to female). The vaginal smears of the females were checked every morning after the start of the mating period to confirm the copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.
Copulation index, fertility index and delivery index was calculated for each group. The calculations were made using the formula as below,
Copulation Index (%) = (No. of rats copulated / No.of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No.of pregnant dams) X 100

Clinical Observations
General clinical observations were made once a day. Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalization, diarrhea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size, Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behavior were recorded.

Functional Observations

Multiple detailed behavioral observations were made in the week before the first treatment and during the last week of the treatment in 5 selected males and on lactation days in 5 selected females (only lactating females were evaluated) of control and treated groups outside the home cage using a functional observational battery of tests :
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioral observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Litter Observations

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
The pre- and post- implantation losses were calculated using number of corpora lutea, number of implantation sites and number of live pups born on PND 0 for each dam. The formula used for the calculation are as follows,
Pre Implantation Loss (%) = (No. of corpora Lutea - No. of implantation site / No. of corpora Lutea) x 100
Post Implantation Loss (%) = (No. of implantation site – No. of live pups / No. of implantation site) x 100
Live pups were counted and sexed and weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattooing. In addition to the observations of parent animals, any abnormal behavior of the offspring was recorded.

Haematology
Haematological parameters from five selected males and females of each group were examined at the end of the treatment.
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined:
Ihaematocrit value (%)
haemoglobin content (g/dL)
red blood cell count (10^12/L)
mean corpuscular volume (fL)
mean corpuscular haemoglobin (pg/erythrocyte)
mean corpuscular haemoglobin concentration (g/dL)
reticulocytes (%)
platelet count (10^9/L)
white blood cells (10^9/L)
neutrophils (%)
lymphocytes (%)
monocytes (%)
eosinophils (%)
basophils (%)

Blood Coagulation

Coagulation parameters from five selected males and females of each group were examined at the end of the treatment.
Blood from the abdominal aorta of the animals was collected in citrate- coated tubes.
The following coagulation parameters were examined:
prothrombin time (sec)
activated partial thromboplastin time (sec)

Clinical Biochemistry
Parameters of clinical biochemistry from five selected males and females of each group were examined at the end of the treatment.
Blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined:
alanine aminotransferase (U/L)
aspartate-aminotransferase (U/L)
alkaline phosphatase (U/L)
creatinine (µmol/L)
total protein (g/L)
albumin (g/L)
urea (mmol/L)
total bile acids (U/L)
total cholesterol (mmol/L)
glucose (mmol/L)
sodium (mmol/L)
potassium (mmol/L)

Urinalysis

A urinalysis was performed with samples collected from 5 selected males of each group at necropsy. Additionally, urine colour/ appearance were recorded.
The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
specific gravity
nitrite
pH-value
protein
glucose
ketone bodies
urobilinogen
bilirubin
blood
leukocytes

Sperm analysis
At necropsy (one day after the last administration) one epididymis and one testis from males of each group were separated and used for evaluation of sperm parameters. Epididymal sperm motility was evaluated in all male animals using Hamilton Thorn Sperm Analyser. The testicular sperm count could not be measured at the end of the study as the testes stored at -80°C were inadvertently taken out of the freezer before the scheduled measurement.

Sacrifice and pathology:
Gross necropsy
All male animals were sacrificed after the completion of the mating period (minimum dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 (maximum dosing period: 54 days) using an anaesthesia was used.
Females showing no sign of pregnancy was sacrificed on day 26 after the last day of the mating period.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weights
The wet weight of the organs of 5 males and 5 females selected from each group was recorded as soon as possible. Paired organs were weighed separately. In addition reproductive organs of all animals were weighed. The enclosed organs were weighed at necropsy:
liver
uterus with cervix
kidneys
thymus
adrenals
thyroid/parathyroid glands
testes
spleen
epididymides
brain
prostate. seminal vesicles and coagulating glands
pituitary gland
ovaries

The following tissues of the same selected animals from each group were preserved in 10 % neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10 % neutral buffered formalin:
brain (cerebrum, cerebellum and pons)
ovaries (females)
spinal cord
uterus with cervix (females)
liver
vagina (females)
kidneys
testes (males)
adrenal glands
epididymides (males)
stomach
prostate and seminal vesicles with coagulating glands as a whole (males)
small and large intestines (including Peyer´s patches)
urinary bladder
thymus
lymphnodes (mesentric and axillary)
thyroid
peripheral nerve (e.g. sciatic nerve) with skeletal muscle
spleen
bone with bone marrow (sternum)
lung and trachea
pituitary gland
mammary glands
oesophagus
heart
gross lesions

Histopathology
All organs and tissues listed above were evaluated from five selected males and females of the control and high dose group:
Reproductive organs and macroscopic changes were evaluated in all study animals. For the testes, a detailed qualitative examination was made.

Statistics:
Evaluation of Results and Statistical Analysis
The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also presented as figures. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and are presented as percentage.
A statistical assessment of the results of the body weight. food consumption, parameters of haematology, blood coagulation and clinical biochemistry, absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
see "Details on results".
Mortality:
no mortality observed
Description (incidence):
see "Details on results".
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see "Details on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see "Details on results".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
see "Details on results".
Haematological findings:
no effects observed
Description (incidence and severity):
see "Details on results".
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see "Details on results".
Urinalysis findings:
no effects observed
Description (incidence and severity):
see "Details on results".
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see "Details on results".
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see "Details on results".
Gross pathological findings:
no effects observed
Description (incidence and severity):
see "Details on results".
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see "Details on results".
Details on results:
Clinical Observation:
There were no clinical signs recorded in male and female animals of treated groups that could be directly related to treatment. However, there were few clinical signs namely moving the bedding, nasal discharge (dark or reddish), salivation and piloerection seen occasionally and transiently during the study period in MD or HD group animals. These findings were considered to be due to local effect but not the systemic effect of the test item. These findings were considered not likely to be adverse. In addition, there was alopecia (on hindlimb, forelimb, thorax and abdominal region) noted in few isolated animals of MD or HD groups, which was assumed to be incidental in origin.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation:
No relevant effects of treatment were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight development:
In both males and females, no adverse treatment related changes were noted for body weight and body weight change during the study period. Statistically there were significant increase in body weight change in female HD group during 2nd week of premating period when compared to control. In addition, there was lower mean body weight gain noted between days 1-7 of premating when compared to control without attaining the statistical significance. But, this increase or decrease in weight gain did not correlate with food intake during the same period. Hence, the changes were not considered likely to be adverse. There was decrease in body weight gain noted in female MD and HD groups during lactation period when compared to control. This decrease had no statistical significance and was not likely to be adverse.

Food Consumption:
In both males and females, no treatment related changes were noted for treated group when compared to corresponding control. The statistical evaluation of the data revealed no significant changes in food intake in treated group animals when compared to control.

Haematology and Coagulation (tables 3 and 4)
No treatment related changes were noted for haematology and blood coagulation parameters measured at the end of treatment period in male and female animals. There were no statistically significant changes noted for haematological and coagulation parameters between the treated and control groups.

Clinical Biochemistry :
There were no treatment related changes considered for measured clinical biochemistry parameters of male and female treated groups when compared to corresponding control. However, there was statistically significant increase noted for mean potassium value in male MD group. In the absence of dose response pattern no relevance to treatment was considered.

Urinalysis:
The urinalysis performed in male animals revealed no considerable changes in treated groups when compared to the control.

Pathology:
At terminal sacrifice, macroscopic organ findings noted were few, and none of them was considered to be test item related.
Yellow spot(s) at the epididymis were observed without dose relationship in 2/10 control males, 1/10 male of LD group, 3/10 males of MD group and 2/10 males of HD group. They were not considered treatment related as histologically they were confirmed to represent spermatic granuloma, a finding known to occur spontaneously in untreated rats of this strain and age. In one isolated male of MD group small testes was noted at necropsy. Histopathologically this finding was considered to be spontaneous in nature and unrelated to the test item.

Organ Weight
There were no changes considered to be related to treatment noted for organ weight in both males and females when compared to corresponding control. However, there was statistically significant increase in relative weight of left kidney weight in male treated (LD, MD and HD) groups, but not total kidney weight. This change in left kidney weight, in the absence of histological changes was not considered to have toxicological relevance.

Histopathology:
Reproductive organs
No test item-related effects were noted on male and female reproductive organs in any of the treatment groups.
Histopathological findings noted in male reproductive organs were few and considered to be spontaneous in nature and unrelated to the test item, comprising multifocal atrophic tubules in the testis of each one male treated at 300 and 1000 mg/kg/day and subsequent epididymal changes in the same animals.
Reproductive organs of most study females showed histomorphological evidence of precedent pregnancy in the uterus. The number of large corpora lutea in the ovary was not essentially different between the groups.
Non pregnant animals showed physiological sexual cycling, and their unsuccessful mating was considered unrelated to the treatment.
Other organs
No test item-related histopathological findings were noted in the other organs evaluated in randomized males and females of the control and high dose group.
Histopathological changes seen at terminal sacrifice were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of the Yttrium Oxide to the male (minimum 28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight/day revealed neither mortalities nor adverse findings of toxicological relevance in male and female animals. There were also no toxicologically relevant findings noted for reproductive and developmental parameters.

Based on the data generated from this “Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test with Yttrium Oxide”, the no observed adverse effect level (NOAEL) for both male and female animals for systemic toxicity was considered to be 1000 mg/ kg body weight/ day.

No classification for repeated-dose toxicity is warranted based on the absence of toxicologically relevant effects in this study, according to the criteria of Annex VI Directive 67/748/EEC or the 1272/2008 regulation -CLP).
Executive summary:

There was no mortality noted in treated (at 100, 300 and 1000 mg/kg bw/day) and control groups during the study period (at least 28 days for males and 54 days for females).

There were few clinical signs namely moving the bedding (1/10 females, HD group), nasal discharge-dark or reddish (3/10 males, HD group; 4/10 females, MD) , salivation (2/10 males, 2/10 females MD; 5/10 females, HD group) and piloerection (1/10 males, 4/10 females MD; 2/10 females HD) seen occasionally and transiently during the study period in MD or HD group animals. These findings were considered to be due to local effect but not the systemic effect of the test item. These findings were considered not likely to be adverse.

During the weekly detailed clinical observation of male and female animals from treated and control groups, no significant changes or differences between the groups were found.

There were no ophthalmoscopic findings in any of the animals of this study.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

In both males and females, no adverse treatment related changes were noted for body weight and body weight change in treated groups when compared to control. There were no treatment related changes noted for food consumption in both male and female treated groups when compared to control.

The statistical evaluation of food consumption revealed no significant changes in food intake in treated group animals when compared to control.

There were no statistically significant changes noted nor for haematological and coagulation parameters nor for clinical biochemistry parameters of male and female treated groups when compared to corresponding control.

The urinalysis performed in male animals revealed no considerable changes in treated groups when compared to the control.

No test item related histopathological findings were seen in the other organs evaluated in selected males and females of the control and high dose group.

At terminal sacrifice, macroscopic organ findings noted were few in both males and females, and none of them was considered to be test item related. There were no changes considered to be related to treatment noted for organ weight in both males and females when compared to corresponding control.