reaction mass of neodymium carbonate and praseodymium carbonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

25-JUL-2008 to 20-MAY-2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline 474 and in compliance GLP. However, as the data are used in a read-across aproach, the maximal reliability score was decreased from 1 to 2 according to Practical Guide n°6.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(see below)

Principles of method if other than guideline:

Deviations:
There was a minor deviation from the study protocol which was not considered to have compromised the validity or integrity of the study in any way: the CPA (positive control) was prepared in water instead of distilled water.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Neodymium oxide

EC Number:

215-214-1

EC Name:

Neodymium oxide

Cas Number:

1313-97-9

IUPAC Name:

neodymium oxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

mouse

Strain:

other: Swiss Ico: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: On the day of treatment, the animals were approximately 6 weeks old.
- Weight at study initiation: At the beginning of treatment the mean bogy weight was 31.5 g for males (ranging from 29.3 to 34.8 g) and 25.7 g for females (ranging from 23.5 to 28.2 g).
- Assigned to test groups randomly: Yes
* Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group.
* Identification: individual tail marking upon treatment.
- Fasting period before study: None
- Housing: The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: All animals had free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany). Each batch of food is analysed by the supplier for composition and contaminant levels.
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analysis of water are performed regularly by external laboratories, These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
- Acclimation period: At least 5 days before the day of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (During the main study the temperature in the animal room was sometimes slightly outside of this range (down to 19°C).
- Humidity (%): 30 to 70 %
- Air changes (per hr): At least 12 cycles/hour of filtered non-recycled fresh air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h (07:00 - 19:00)

IN-LIFE DATES: From 04-NOV-2008 to 06-NOV-2008

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Name: 0.9% NaCl
- Batch Nos.: WGP092/6 and WGP092/7
- Supplier: Laboratoire Fresenius Kabi, Sèvres, France.
- Route and frequency of administration: Intraperitoneal / two treatments separated by 24 hours
- Volume of administration: 10 mL/kg

Details on exposure:

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.
In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given intraperitoneal administrations of the test material at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 1000 and 2000 mg/kg/day for males and females respectively, over a 2-day period.
One group of five males and five females received the vehicle (NaCl 0.9%) under the same experimental conditions, and acted as control group.
One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Two treatments separated by 24 hours

Post exposure period:

Not included

Doses / concentrations

Remarks:

Doses / Concentrations:
187.5, 375 and 750 mg/kg/day and 500, 1000 and 2000 mg/kg/day for males and females, respectively
Basis:
nominal conc.

No. of animals per sex per dose:

Preliminary toxicity test: 9 male and 12 female mice were used.
Main cytogenetic test: 56 mice; 28 males and 28 females were used, i.e. 5 animals per sex per dose level.
Blood sampling: 6 mice; 3 males at dose-level 750 mg/kg/day and 3 females at dose-level 2000 mg/kg/day were used.

Control animals:

yes, concurrent vehicle

Positive control(s):

- The positive control was Cyclophosphamide (CPA, CAS No. 50-18-0, Sigma, Saint-Quentin-Fallavier, France), batch No. 076K1050 dissolved in distilled water at a concentration of 50 mg/kg. The preparation was stored at -20°C and thawed immediately before use.
- Route and frequency of administration: Oral / one treatment
- Volume of administration: 10 mL/kg

Examinations

Tissues and cell types examined:

Erythrocytes in the bone marrow of mice

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.
In order to select the top dose-level for the cytogenetic study, 500, 750 and 1000 mg/kg were administered twice, 24 hours apart, to three males and 500, 1000, 1500 and 2000 mg/kg were administered twice, 24 hours apart, to three females.

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines:
- since toxic effects were observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality for males;
- since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day for females.

TREATMENT AND SAMPLING TIMES:
In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given intraperitoneal administrations of the test material at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 100 and 2000 mg/kg/day for males and females respectively, over a 2-day period.
One group of five males and five females received the vehicle (NaCl 0.9%) under the same experimental conditions, and acted as control group.
One group of five males and five females received the positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.
Blood samples for the determination of plasma levels of the test material were taken at the following time: 1 hour (three males and three females) following the second treatment with the high dose level.

DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, all the animals were deeply anaesthetised by an intraperitoneal injection of sodium pentobarbital, then killed by exsanguination. The femurs were removed and the bone marrow was flushed out with foetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were re-suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa.
The slides were coded so that the scorer was unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the slides was performed at Microptic, cytogenetic services, 2 Langland Close Mumbles, Swansea SA3 4LY, UK, in compliance with GLP.

OTHER:
Blood samples for the determination of plasma levels of the test material were taken at the following time: 1 hour (three males and three females) following the second treatment with the high dose level.
Venous blood (approximately 1 mL) was taken into a tube containing lithium heparinate from the orbital sinus of the animals under light isoflurane anaesthesia.
After the blood sampling, the animals were deeply anaesthetised by an intraperitoneal injection of sodium pentobarbital, then killed by exsanguination and discarded without necropsy.The blood was centrifuged (10 min at 4000 rpm, at +4°C) and the plasma was kept frozen in individual tubes at -20°C.

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

Normality and homogeneity of variances has been tested using a Kolmogorov Smirnov test and a Bartlett test.If normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (> or = three groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (> or = three groups) was performed followed by a Dunn test (if necessary).All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A decrease in the PE/NE ratios was observed in males and females at all tested dose-levels compared to the vehicle control.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
At 500 mg/kg/day (three males and three females), neither mortality nor clinical signs were noted in any animals.
At 750 mg/kg/day (three males), no mortality occurred, one out of the three males showed piloerection.
At 1000 mg/kg/day (three males and three females), one out of three males was found dead 24 hours after the second treatment. Pilorection, hypoactivity and half-closed eyes were noted prior to its death. Surviving animals (including females) showed piloerection and hypoactivity.
At 1500 mg/kg/day (three females), no mortality occurred, one out of three females showed piloerection.
At 2000 mg/kg/day (three females), no mortality occurred, all females showed piloerection, together with half-closed eyes for one animal.
No change in the body temperature which could be considered as excessive (increase of at least 1°C or decrease of at least 3°C for 5 or more hours) was recorded in the animals following either treatment, at any tested dose-levels.

RESULTS OF DEFINITIVE STUDY
No clinical signs and no mortality were observed in males given 187.5 mg/kg/day and in females given, 500, 1000 and 2000 mg/kg/day.
At 375 mg/kg/day (five males) and 750 mg/kg/day (eight males), no mortality occurred, but piloerection was observed following the second treatment in 2/5 males and 7/8 males, respectively.
The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with historical data. Cyclophosphamide induced significant increases (p<0.05 in males and p<0.01 in females) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered as valid.
The mean MPE values in the test material treated groups were similar to those of the vehicle group.
A decrease in the PE/NE ratios was observed at all tested dose-levels compared to the vehicle control. In females, this decrease was statistically significant (up to p<0.01), showing that the bone marrow cells were effectively exposed to the test material.
The determination of the plasma levels of the test material showed that at least 1/3 males and 2/3 females were exposed to the test material, since the concentrations in the samples taken 1 hour following the second treatment were 4.84, 12.1 and 14.7 ng/g, respectively.
Taking into account the clinical signs observed in some animals from the high-dose group, the test material concentrations found in the plasma samples and finally the decrease in the PE/NE ratios, the systemic exposure of the animals as well as the exposure of the bone marrow cells to the test material were clearly demonstrated.

Any other information on results incl. tables

Results of the cytogenetic test :

	Group	Doses     (mg/kg/day)	MPE/1000PE		PE/NE ratio		Time of sacrifice after the last administration
			mean	(sd)	mean	(sd)
Males							24 h
	Vehicle	-	1.7	(0.9)	0.8	(0.2)
		187.5	1.9	(1.1)	0.4	(0.2)
	Test material	375	1.0	(0.9)	0.4	(0.1)
		750	1.7	(1.0)	0.3	(0.1)
	Cyclophosphamide	50	25.1	(5.5)*	1.0	(0.1)
Females							24 h
	Vehicle	-	1.2	(0.4)	0.7	(0.1)
		500	0.6	(0.7)	0.4	(0.1)
	Test material	1000	1.2	(0.7)	0.3	(0.2)**
		2000	0.9	(1.2)	0.4	(0.2)*
	Cyclophosphamide	50	17.5	(4.4)**	0.7	(0.2)

Five animals per group

MPE : Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

sd: standard deviation

 

Statistical significance:

* p <0.05

** p <0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions of this study, the test material, neodymium oxide, did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 187.5, 375 and 750 mg/kg/day in males or at the dose-levels of 500, 1000 and 2000 mg/kg/day in females.

Executive summary:

The potential of the test material, neodymium oxide, to induce structural or numerical damage in bone marrow cells of Swiss Ico: OF1 (IOPS Caw) mice was evaluated in a study performed according to the standardised guidelines OECD 474 and EU Method B.12 and in compliance with GLP.

 

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female mice were administered the test material via the intraperitoneal route at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 100 and 2000 mg/kg/day for males and females respectively, over a 2-day period.

Three additional male and female mice were added in the high dose groups in order to determine the plasma level of the test material. These animals were killed 1 hour after the last treatment and their blood collected, centrifuged and frozen until determination of the test material level in the plasma.

An additional group received the vehicle under the same experimental conditions and acted as control and another group received a positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

 

No clinical signs and no mortality were observed in males given 187.5 mg/kg/day and in females given 500, 1000 and 2000 mg/kg/day. At 375 and 750 mg/kg/day (male groups), no mortality occurred, but piloerection was observed in some animals following the second treatment. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with historical data. The study was therefore considered valid.

The mean MPE values in the test material treated groups were similar to those of the vehicle group indicating no effect of the test material.

A decrease in the PE/NE ratios was observed at all tested dose-levels compared to the vehicle control. In females, this decrease was statistically significant (up to p < 0.01), showing that the bone marrow cells were effectively exposed to the test material.

The determination of the plasma levels of the test material showed that at least 1/3 males and 2/3 females were exposed to the test material, since the concentrations in the samples taken 1 hour following the second treatment were 4.84, 12.1 and 14.7 ng/g, respectively.

Taking into account the clinical signs observed in some animals from the high-dose group, the test material concentrations found in the plasma samples and finally the decrease in the PE/NE ratios, the systemic exposure of the animals, as well as the exposure of the bone marrow cells to the test material, were clearly demonstrated.

 

Under the conditions of this experiment, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells.