Yttrium oxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1st october 2012 - 14th april 2014

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 for cross-reference:

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

Guideline:

other: according to 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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1214-36-9

IUPAC Name:

1214-36-9

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

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 ofthe 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 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)

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 in 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 most recently measured on week- based.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analyzed for nominal concentration. 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.
The analysis was done by ICP-OES (inductively coupled plasma with optical emission spectrometry).

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.

Duration of treatment / 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 in 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 most recently measured on week- based.

Frequency of treatment:

Once daily

Duration of test:

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 and the rest 5 animals from each group received dose administration for 29 days) was completed.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Doses / concentrations 4

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

Maternal 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.
Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and macroscopic changes were evaluated in all study animals (also see section 7.8.1 Toxicity to reproduction).
Other parameters were observed and have been detailled under section 7.5.1 Repeated Dose Toxicity - oral : clinical and functional observations, pathology, histopathology, haemotology, clinical biochemistry, blood coagulation, urinalysis.

Ovaries and uterine content:

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.

Fetal examinations:

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. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Implantation losses (pre and post) and reproductive indices (copulation, fertility and delivery) were calculated based on the data obtained.

Pups sacrificed on post natal day 4 were carefully examined for gross external 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.

Statistics:

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 and reproductive and development parameters were performed by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test (a normal distribution was assumed based on the in vivo data). These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Indices:

Reproductive indices measured:
- Copulation index (%), fertility index (%),
- Delivery index (%)
- Viability index (%)

Index related to litter:
Number of corpora lutea, number of implantation sites and number of live pups born on PND 0 for each dam were evaluated.
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
Number of Live and sexed pups
Weighed of pups within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical observations:
There were no clinical signs recorded of treated groups that could be directly related to treatment.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

Functional Observations:
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:
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.
One female from medium dose (No. 68) did not show the evidence of mating through vaginal smear. However, animal was pregnant and littered. Hence, the body weight data measured during the gestation period is excluded as the precise GD 0 was not clear for this animal.

Food Consumption
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.
One female from medium dose did not show evidence of mating through vaginal smear. However, animal was pregnant and littered. Hence, the food consumption data measured during the gestation period is excluded as the precise GD 0 was not clear for this animal.

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

Precoital Interval and Duration of Gestation
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.

Reproductive Indices (table 3)
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 in 100% pregnancy rates in C and HD groups and 90% pregnancy rates in LD and MD groups. One female of LD group and one female of MD group were found not to be pregnant at terminal sacrifice.
Histologically, they showed physiological sexual cycling, and their unsuccessful mating was considered unrelated to the treatment.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data (table 1)
No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4. Statistical evaluation of data revealed no significant differences between the values of treated and control groups.

Litter Weight Data
No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control. The statistical evaluation of data revealed no significant differences between the values of the treated and control groups.

Pre- and Post-Natal Data (Table 2)
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.

Pup Survival Data (Table 4)
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, LD and HD 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 (%).

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1 : Litter Data

											PND 0												PND 4
Total No. of Pups			No. of Male			No. of Fem.			Sex Ratio (m/f)			Live Pups			Still Birth				Runt			No. of Male			No. of Fem.			Live Pups			Sex Ratio (m/f)
C
Mean		11.1			5.1			6.0			1.0			10.9			0.2			0.0			5.1			5.7			10.8			1.0
SD		3.21			2.42			2.21			0.63			3.18			0.42			0.00			2.42			2.36			3.46			0.61
N		10			10			10			10			10			10			10			10			10			10			10
LD
Mean		11.0			5.6			5.4			1.2			11.0			0.0			0.0			5.6			5.3			10.9			1.2
SD		2.12			1.81			1.81			0.81			2.12			0.00			0.00			1.81			1.73			2.03			0.80
N		9			9			9			9			9			9			9			9			9			9			9
MD
Mean		11.1			5.1			6.0			1.2			11.1			0.0			0.0			5.1			6.0			11.1			1.2
SD		1.76			2.26			2.45			1.22			1.76			0.00			0.00			2.26			2.45			1.76			1.22
N		9			9			9			9			9			9			9			9			9			9			9
HD
Mean		11.8			4.7			7.1			0.7			11.5			0.3			0.0			4.5			6.9			11.4			0.7
SD		1.48			1.34			1.37			0.32			1.72			0.95			0.00			1.27			1.45			1.65			0.30
N		10			10			10			10			10			10			10			10			10			10			10

Table 2 :Pre- and Post-Natal Data

Group		No. Corpora Lutea (CL)		No. Implantation Sites (IS)		No. Live Pups on PND 0		No. Live Pups on PND 4		Pre Implantation Loss (%)		Post Implantation Loss (%)
C
Mean		12.50		11.60		10.90		10.80		6.64		7.24
SD		3.37		3.06		3.18		3.46		5.03		8.87
N		10		10		10		10		10		10
LD
Mean		12.44		11.11		11.00		10.89		10.71		1.01
SD		1.88		2.09		2.12		2.03		10.26		3.03
N		9		9		9		9		9		9
MD
Mean		13.33		11.78		11.11		11.11		9.73		5.65
SD		3.24		1.72		1.76		1.76		10.90		6.29
N		9		9		9		9		9		9
HD
Mean		12.80		12.10		11.50		11.40		4.77		4.92
SD		2.10		1.60		1.72		1.65		7.20		7.23
N		10		10		10		10		10		10

 

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

Table 3 :Mean Reproductive Indices

Parameters		C		LD		MD		HD
Copulation Index (%)		100		100		100		100
Fertility Index (%)		100		90		90		100
Delivery Index(%)		100		100		100		100
Viability Index (%)
Mean		95		99		100		97
SD		10.5		2.50		0.0		8.0
N		10		9		9		10

Corpulation 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

 

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 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 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 animals (males and females) and pups for systemic toxicity was considered to be 1000 mg/ kg body weight/ day. The no observed effect level (NOEL) for the reproductive and developmental toxicity was considered to be 1000 mg/kg body weight/day.

Executive summary:

The aim of this study was to assess the possible effects of Yttrium Oxide on male and female fertility and embryofetal development including the oral toxicity of Yttrium Oxide after repeated dose administration by oral gavage. This study was performed according to OECD guideline 422 and in compliance with GLP.

The test item was administered daily by oral gavage in graduated doses to 3 groups of test animals, one dose level per group for a treatment period maximum of 54 days in females and minimum 28 days in males. Animals of control group were handled identically as the dose groups but received same dose volume of the vehicle (aqueous carboxymethylcellulose solution 0.5% w/w) used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

The following doses were evaluated:

Control (C): 0 mg/kg body weight/ day

Low Dose (LD): 100 mg/kg body weight/ day

Medium Dose (MD): 300 mg/kg body weight/ day

High Dose (HD): 1000 mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was suspended in 0.5% aqueous carboxy methylcellulose and administered daily during 14 days of pre-mating and maximum 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for minimum of 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

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

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 from 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.

No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio and live pups on PND 0 and PND 4.

No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control.

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.

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.

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

No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in treated group when compared to controls. No treatment related changes were considered for viability index (%).

No treatment related gross external findings were observed in the pups of treated groups.

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 animals (males and females) and pups for systemic toxicity was considered to be 1000 mg/ kg body weight/ day. The no observed effect level (NOEL) for the reproductive and developmental toxicity was considered to be 1000 mg/kg body weight/day.