Registration Dossier

Administrative data

Endpoint:
neurotoxicity, other
Remarks:
developmental neurotoxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Neurobehavioral Assessment of Rats Exposed to Yttrium Nitrate during Development
Author:
Li CX, Ma C, Fang HQ, Zhi Y, Yu Z, Xu HB, Jia XD
Year:
2015
Bibliographic source:
Biomedical and Environmental Sciences 28, 281-290

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
- The test material was prepared by dissolving yttrium oxide (purity > 99.99%) in nitric acid and diluting it to a pH of 6 using 1 M NaOH and deionised water.
- Source of Y2O3: Beijing Research Institute of Nonferrous Metals, Beijing, China

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China)
- Age at study initiation: Sexually mature; no further specifications
- Sex: Virgin female and male rats
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing: Not specified; pregnant rats housed singly during dosing
- Diet (e.g. ad libitum): Standard rat chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40%-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
gestation day (GD) 6 to postnatal day (PND) 21 (28 days)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
referring to aqueous solution of yttrium trinitrate
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
referring to aqueous solution of yttrium trinitrate
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
referring to aqueous solution of yttrium trinitrate
Dose / conc.:
45 mg/kg bw/day (nominal)
Remarks:
referring to aqueous solution of yttrium trinitrate
No. of animals per sex per dose:
4 groups of 20 females each
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Not specified
- Mating procedure:
*After 2 weeks of acclimatisation each female rat was mated with a resident male of the same strain.
*Proof of pregnancy: Evidence of a vaginal plug was used to indicate successful mating and Day 0 of pregnancy.
- Rationale for animal assignment (if not random): Random
- Pregnant females were allowed to deliver their pups spontaneously and nurse them.
- On postnatal day 4, the number of pups in each litter was reduced to 8 (4 males and 4 females). The pattern of pup assignment was based on OECD guideline 426, with slight modifications.

Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0, 3, 7, 10, 14, 17, 21 and postnatal day 1, 4, 7, 10, 14, 17, 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice of dams on postnatal day 21.
- The dams were subjected to a gross examination (not further specified).
- Pregnancy status was determined for those rats that failed to deliver.

OTHER (observations in pups): Yes
- Each litter was observed daily for signs of toxicity.
- Body weight: postnatal day 1, 4, 7, 10, 14, 17, 21 and weekly thereafter until postnatal day 70
- Food consumption was calculated weekly from postnatal day 21 to 70.
- On postnatal day 1, the number of live and dead pups was determined and the sex ratio of the pups was recorded.
- The first set of pups (20 per sex and dose level; 1 male and 1 female per litter) was evaluated for pre-weaning behaviour using behavioural ontogeny tests and for histopathology.
- Following weaning on postnatal day 21, half of the pups (10 per sex and dose level) were sacrificed and their brains were removed, weighed, and frozen for further research. The other half of the pups (n=10) were anesthetised using pentobarbital sodium and perfused with 4% paraformaldehyde. Their brains were then removed and weighed.
- The second set of pups was evaluated for locomotor activity on postnatal day 13, 17, 21 and 67 and subjected to the rotating rod test (postnatal day 25) and the hot plate test (postnatal day 24 and 61). Sexual maturation was also determined.
- From this group, 10 pups per gender and dose level were anesthetised and fixed using a perfusion of 4% paraformaldehyde on postnatal day 70. Their brains were then removed, weighed, and processed for neuropathological examination.
- A third set of pups was subjected to the Morris water maze test. Half of them were tested on postnatal day 25 and the other half on postnatal day 62.
- From this group, on postnatal day 70, 10 males and 10 females were sacrificed. Their brains were also removed and weighed.
- The fourth set of pups was for another experiment. Half of them were exposed to yttrium trinitrate by gavage from postnatal day 21 until postnatal day 70.
- Remaining pups were sacrificed.
Specific biochemical examinations:
None
Neurobehavioural examinations performed and frequency:
Postnatal Development of Offspring
Postnatal neurobehavioural development was evaluated beginning PND 4 by observing the surface-righting reflex on PND 4, cliff avoidance on PND 6, forelimb grip strength on PND 12, startle response on PND 13, homing reflex on PND 13, and air-righting reflex on PND 17, testes descent on PND 27, and vaginal opening on PND 31.

Locomotor Activity
Locomotor activity was assessed on PND 13, 17, 21, and 67 using the JLBehv animal locomotion analysis system (Jiling Software Technology Co, Ltd; Shanghai, China). Each rat was tested for 6 min in a plastic box surrounded by black plastic enclosures to decrease the risk of distractions in the external environment.

Morris Water Maze Test
To assess hippocampus-dependent spatial learning and memory, pups were tested in the Morris water maze on PND 25 or PND 62 with slight modifications. Briefly, a black circular pool (150 cm in diameter and 50 cm in height) filled with water (23°C) was placed in the experimental room with fixed visual clues on walls. The pool was divided into four quadrants, and an escape black platform (diameter: 11 cm) was placed 1 cm below the water surface in the middle of one of the quadrants. The position of the platform was not changed throughout the experiment. Rats were trained in three trials daily over 5 consecutive days. In each trial, the rats were placed in the water facing the wall at one of the three starting positions (not the platform quadrant) and were permitted to swim until they reached the platform. If a rat failed to find the platform within 60 s, it was guided manually toward the platform by the researcher for 15 s. Each trial was recorded using a video tracking system; the escape latency and distance were measured using SMG-2 software (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College). For the probe trial, the hidden platform was removed. The animal was then released at the same starting position and allowed to swim freely for 60 s. The time and distance crossed in the target quadrant were recorded. For the visible platform task, performed on the final day of the test, the platform was placed so that it protruded 2 cm above the surface of the water.

Rota-rod Test
This test is designed to evaluate coordination and balance. It was carried out by placing a rat on a rotating drum and measuring the length of time the animal was able to maintain its balance while walking on top of a rod that sat 15 cm above the base. The rod was divided into five equal segments by six disks. The animals were trained before being tested by being placed on the rod while it rotated at a speed of 20 rpm. The length of time the rat could stay on the rod without falling was recorded. The test was repeated three times at 30-min intervals on PND 25. Each rat was allowed to stay on the rod a maximum of 300 s.

Hot Plate
This test was carried out to evaluate nociception. The rat was placed on a hot-plate apparatus that was maintained at 52°C. The time until the rat lifted or licked a paw was recorded. The measurement was repeated three times on PND 24 and on PND 61, and the average of the three values was used as the baseline thermal nociceptive threshold.
Sacrifice and (histo)pathology:
- Sacrifice of dams on PND 21 - no histopathological examination performed.
- Set 1 of pups - sacrifice on PND 21 - brains removed and weighed.
- Set 2 and 3 of pups - sacrifice on PND 70 - brains removed and weighed.
Other examinations:
Postnatal survival, mean number of pups, percentage of males per litter, average pup weight, PND of testes descent or vaginal opening.
Statistics:
The Morris water maze test results were analysed by a multivariate analysis of variance (ANOVA) of the general linear model (GLM) to determine the effects of between-subject factors (group), within-subject factors (day), and group by day of interaction on the dependent variables. Group differences on separate acquisition days, the effects on probe trial tests and the visible platform task, as well as body weight, food consumption data, and other behavioural test results were analysed using one-way ANOVA. The significance of intergroup differences was analysed using a two-sided Dunnett test. Significance was defined by P value <0.05, using SPSS 11.5 software.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or toxic effects that could be attributed to the administration of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality observed among the maternal animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences observed between the (maternal) animal groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Similar among all (maternal) animals during gestation and lactation.
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):
no effects observed
Description (incidence and severity):
- Pre-weaning behavioural tests: No effects of toxicological relevance related to treatment were observed.
- Rota-rod and hot plate test: There were no significant differences among groups of pups in either gender on the Rota-rod tests and hot plate tests.
- Locomotor activity test: No effects of toxicological relevance related to treatment were observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Effects observed on brain weight were not considered related to maternal test item administration due to the lack of a dose-response relationship.
Gross pathological findings:
no effects observed
Description (incidence and severity):
An evaluation of rats that failed to deliver revealed no implantation sites, indicating a negative pregnancy status for each animal.
No further results reported.
Neuropathological findings:
no effects observed
Description (incidence and severity):
In the Morris water maze test there was no significant difference or trend between groups during the learning and memory trials, which started on PND 25 and 62, respectively.
Considering spatial reference memory (tested on PND 6), no differences were observed in terms of the time spent in the quadrant in which the platform was previously located (assessed using the probe trial).
All groups were subjected to a test of their ability to escape to a visible platform, but the difference in findings was nog significant.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Postnatal survival, mean number of pups, percentage of males per litter, average pup weight, PND of testes descent or vaginal opening, were not affected at any dose level. Changes in food consumption did not reveal a dose-response relationship and were not considered related to yttrium exposure.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 45 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

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this developmental neurotoxicity study, female rats were exposed from gestation day 6 onwards to postnatal day 21 to 5, 15 or 45 mg/kg bw/day of an yttrium trinitrate aqueous solution. The pups were followed up to postnatal day 70. No toxicologically relevant effects related to the treatment have been observed in maternal animals or their offspring over the course of the study. The NOAEL was therefore considered to be higher than or equal to 45 mg/kg bw/day, i.e. the highest dose tested. Based on the results of this study, there are no indications for yttrium (administered as yttrium trinitrate) to be a developmental neurotoxicant.