Yttrium

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Yttrium oxide

EC Number:

215-233-5

EC Name:

Yttrium oxide

Cas Number:

1314-36-9

Molecular formula:

Y2O3

IUPAC Name:

Yttrium(III) oxide

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)
Colour: white

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC 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 exposure:

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 study 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.

Details on mating procedure:

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.

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.

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 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 was completed.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

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

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

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 Sperm Analyser. The testicular sperm count could not be measured at the end of the study as the testes stored at -80oC were inadvertently taken out of the freezer before the scheduled measurement.

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 behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

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. Pups were killed on PND 4 by using high dose of sodium pentobarbitol.
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

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.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

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 were evaluated from five selected males and females of the control and high dose group:
Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and macroscopic changes were evaluated in all study animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Postmortem examinations (offspring):

Pups were killed on PND 4 by using high dose of sodium pentobarbitol.
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

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 and 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).

Reproductive indices:

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 pregant / No.of females copulated) X 100

Offspring viability indices:

Delivery Index (%) = (No. of dams with live newborns / No.of pregnant dams) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

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 transciently 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) of 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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In both males and females, no 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 and compound intake (if feeding study):

no effects observed

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Pre- and Post-Natal Data (Tables 2 and 3)
No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre and post implantation loss in treated groups when compared to control. The statistical evaluation of data revealed no significant differences between the values of treated and control groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm analysis (table 1):
There were no treatment related changes noted for epididymal sperm motility (Motile Count %, Static Count % and Rapid Count %) measured in all animals of treated and control groups. The statistical analysis of epididymal sperm motility indicated no statistical significant difference between the treated and control groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre- and Post-Natal Data (Tables 2 and 3)
No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre and post implantation loss in treated groups when compared to control. The statistical evaluation of data revealed no significant differences between the values of treated and control groups.

Details on results (P0)

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.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Pup Survival Data (table 5)
No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in treated groups when compared to control. However, there was 1 pup each in Control (Pup no. 3; animal 50), LD (Pup no. 12, animal 58) and MD (Pup no. 9, animal 70) groups found dead or missing between PND 0 and 4. The missing pup was assumed to be cannibalized by dam, which was considered to be incidental. No treatment related changes were considered for viability index (%).

Body weight and weight changes:

no effects observed

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related gross external findings were observed in any of the treated groups. However, there were few isolated findings noted in pups namely dry skin, dark spot on/ or dark abdomen, dark snout in control group; dark spot on forelimb and thoracic back, dark snout in LD group; dry skin in MD and HD groups. These findings were considered to be incidental in origin.

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

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:

In conclusion, the repeated dose administration of yttrium oxide to male (minimum 28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor 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 results, the no observed adverse effect level (NOAEL) for systemic and reproductive and developmental toxicity is considered to be 1000 mg/kg body weight. The NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day and the NOEL for toxic effects on progeny was 1000 mg/kg/day. Yttrium oxide is considered not classified as reproductive toxicant according to the CLP regulation.

Executive summary:

The statistical analysis of epididymal sperm motility indicated no statistical significant difference between the treated and control groups.

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.

No test item-related effects were noted on male and female reproductive organs in any of the treatment groups.

No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control. All pregnancies resulted in normal births.

There were no treatment related changes noted for copulation index (%), fertility index (%), delivery index (%) and viability index (%) in treated groups when compared to corresponding control group.

Successful mating resulted 100% pregnancy rates in C and HD groups and 90% pregnancy rates in LD and MD groups.

Histologically, they showed physiological sexual cycling, and their unsuccessful mating was considered unrelated to the treatment.

The statistical evaluation of pre and post-natal data revealed no significant differences between the values of treated and control groups.

No treatment related no significant changes were noted for survival of the pups and for viability index (%).