Praseodymium(III,IV) oxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2018 to 30 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

transgenic rodent mutagenicity assay

Test material

Test material information

Constituent 1

Reference substance name:

Praseodymium(III,IV) oxide

EC Number:

234-857-9

EC Name:

Praseodymium(III,IV) oxide

Cas Number:

12037-29-5

Molecular formula:

O11Pr6

IUPAC Name:

tetrakis(λ⁴-praseodymium(4+)) dipraseodymium(3+) undecaoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Storage Conditions: Under room temperature (1 to 30 °C) in the dark and sealed container.
- Stability: Stable under the specified condition until the expiration date (26 September 2019).

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Under room temperature in the dark and sealed container
- Stability under test conditions: Stable

Test animals

Species:

mouse

Strain:

other: CD2-LacZ80/HazfBR (Muta™Mouse) [SPF]

Remarks:

Transgenic mouse

Details on species / strain selection:

CD2-LacZ80/HazfBR mice are commonly used as transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks of age at the time of assignment to groups.
- Weight at study initiation: 23.6 to 27.3 g
- Assigned to test groups randomly: Animals were assigned to groups based on their body weights on Day 1 using LATOX-F/V5 computer system package. Unassigned animals were excluded from the study on Day 1. The weight range of animals was within the mean weight ± 20 %.
- Housing: All animals were housed in a barrier system room under positive pressure. Two or three animals were housed in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding. Analytical results of levels of contaminants in the bedding were within the acceptable limits of the Japan Experimental Animal Feed Association. The animal cages, the beddings and the feeders were replaced on the day of grouping and once a week thereafter. The water bottles were replaced once every 2 or 3 days.
- Diet: Ad libitum access to pellet diet CRF-1. Food was replenished at the time of exchanging the feeders. Contaminant levels were within the acceptable limits proposed by the Japan Experimental Animal Feed Association.
- Water: Animals were provided access to tap water from water bottles ad libitum. Contaminant levels in the water were within the acceptable limits of the Tap Water Quality Standard. Examination also confirmed that no bacteria were detected in the water.
- Acclimation period: Upon arrival, each animal was monitored for their general condition and body weight gain. During the quarantine and acclimation period (Day -8 to Day 1), the animals were observed once a day. The body weight of each animal was measured on the day of receipt and at the end of the quarantine and acclimation period. None of the animals showed abnormalities in their general condition or body weight gain.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26 °C (actual values: 22.8 to 23.0 °C)
- Humidity (%): 35 to 70 %RH (actual values: 40 to 55 %RH)
- Air changes (per hr): 12 times or more/hour
- Photoperiod (hrs dark / hrs light): 12 hours (light on: 7:00, light off: 19:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Methylcellulose 400
- Lot/batch no.: WDP4521
- 0.5 % (w/v) MC, the vehicle to prepare test material formulations, was used as a negative control.
- Dose volume: 10 mL/kg
- Preparation of vehicle: An aqueous solution of methylcellulose 400 at 0.5 % (w/v) was used for the negative control. Methylcellulose 400 (4 g) was accurately weighed and transferred into a stoppered volumetric cylinder. A small volume of warmed (approximately 80 °C) water for injection was repeatedly added to dissolve it. After this solution was cooled to room temperature, it was adjusted to 800 mL with water for injection to make 0.5 w/v% solution. The prepared vehicle was stored in a refrigerator and was used within 14 days after preparation.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- 2.5, 5.0 or 10 g of the test material for the 25, 50 and 100 mg/mL solutions respectively were weighed and transferred to a stoppered measuring cylinder and mixed with a little vehicle.
- Solutions were diluted to 100 mL with vehicle to prepare 25, 50 and 100 mg/mL formulations, respectively.
- The formulations were fully mixed by manual inversion and then stirred by magnetic stirrer until visibly homogeneous.

Within a concentration range of 10.0 to 200 mg/mL, the test material was confirmed to be stable and homogeneous in 0.5 % (w/v) methylcellulose 400 after storage for at least 8 days at ambient laboratory temperature and 2 to 8 °C in the dark (non-GLP validation study). In the same non-GLP validation study it was confirmed under non-GLP condition that the 10.0 and 100 mg/mL dosing formulations stored at 0 to 10 °C were stable and homogeneous in 0.5 w/v% MC for at least 15 days.

Dispensing and storage of the test material formulations
- Each test material formulation was divided into 5 or 7 portions (including 1 portion as reserve) for each administration under continuous stirring using a magnetic stirrer and stored in tight and light-resistant containers in a refrigerator (acceptable temperature range: 1 to 9 °C) until use. The actual temperature range was 3.6 to 7.6 °C during the storage period. The stored formulations were fully mixed by manual inversion and then stirred by a magnetic stirrer at least 30 minutes before use.

Analysis of test material formulations
- The analysis for the test material formulations was conducted at IDEA Consultants, Inc. under non-GLP condition.
- A portion (approximately 6 mL) of the test material formulations prepared on the first preparation day was submitted to IDEA Consultants, Inc. The concentration of each sample was measured using ICP-MS and the ratio to the nominal concentration was calculated from the mean concentrations. If the ratios of the mean concentration of the test material in each formulations to the nominal concentrations are within 10 % and the relative standard deviation (RSD) of the concentrations in three layers is 10 % or less, the test material formulations were judged to be appropriately prepared.
- As a result, the ratios to the nominal concentrations in the test material formulations were 108.0 to 109.8 %, and the RSD were 3.8 to 5.2 %. These results showed that the test material formulations had been appropriately prepared.

RATIONALE FOR SELECTION OF THE DOSE LEVEL
- In the acute oral toxicity study male and female Sprague-Dawley (SD) rats treated with 2000 mg/kg of test material in 0.5 % (w/v) methylcellulose, no clinical signs and no changes of the body weight gain of the animals were observed. Combined repeated dose and reproduction/developmental screening study using SD rats revealed that the no observed adverse effect level (NOAEL) was 1000 mg/kg/day.
-Toxicity to mice was not observed, in the dose range-finding study in CD2F1/Slc mice treated with 100, 300 or 1000 mg/kg of test material in 0.5 w/v% MC. Therefore, a dosage of 1000 mg/kg/day was selected as the high dose and 2 dosage levels including 250 and 500 mg/kg/day as lower doses were used in the present study.

ADMINISTRATION METHOD
- Before administration, the test material formulations were shaken by hand. Then, the individual dosing volumes of test material formulations were taken under continuous stirring using a magnetic stirrer for at least 30 minutes before use.
- The vehicle and the test material were administered orally according to the test guideline.
- The vehicle and the test material were administered to animals once daily for 28 consecutive days by using a disposable syringe with a Teflon sonde.
- The dosing volume (mL) was set at 0.1 mL per 10 g of body weight. The individual dosing volume (mL) was calculated on the basis of the most recent individual body weight

Duration of treatment / exposure:

- The administration period was 4 weeks (28 days).
- To ensure that the data would be available for each 5 animals in a group for the main study and germ cell study, 12 animals each were assigned in all groups. At first, 5 animals were selected by the ascending order of animal ID No. and used for evaluation in the main study. The remaining animals were retained for approximately 7 weeks after the end of the dosing period in order to collect germ cells, for retention as a contigency in the event that germ cell assessments were necessary.

Frequency of treatment:

Once daily

Post exposure period:

Manifestation time 3 days for animals assigned to somatic tissue assessments (main study); to enable adequate sperm maturation animals assigned to germ cell analysis had a manifestation time of 7 weeks (49 days).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day

Remarks:

Concentration of dosing formulation: 25 mg/mL

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

Concentration of dosing formulation: 50 mg/mL

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Concentration of dosing formulation: 100 mg/mL

No. of animals per sex per dose:

5 male mice per group in the main study.
7 male mice per group in the germ cell study.

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

N-ethyl-N-nitrosourea (ENU), contains 40 % water, 1.8 % AcOH.
- Justification for choice of positive control(s): Based on consideration of information in academic documents.

PREPARATION OF POSITIVE CONTROL SOLUTION
- 50 mg of ENU was weighed, transferred into a graduated test tube and diluted to 5 mL with 1/15 mol/L sodium-phosphate buffer (pH 6) to make a 10 mg/mL solution. The positive control solution was prepared just before use.
- The dose was 100 mg/kg.
- Concentration of dosing formulation: 10 mg/mL
- Dosing volume: 10 mL/kg
- The positive control material was administered intraperitoneally once daily for 2 consecutive days at about 24-hour intervals using a disposable syringe attaching a 25G needle. The dosing volume was set at 0.1 mL per 10 g of body weight and was calculated on the basis of the most recent individual body weight

Examinations

Tissues and cell types examined:

Main study: The liver, stomach, testes and vas deferens/cauda epididymis were removed from each animal.
Germ cell study: The testes and cauda epididymis were separately removed from each animal.

Details of tissue and slide preparation:

TISSUE RETENTION
LIVER: Two samples were prepared from the left lateral lobe, Each sample was separately put into microtubes and frozen in liquid nitrogen (LN2). The other lobes were put into a storage bag, squashed and then frozen in LN2. The remaining part of left lateral lobe was fixed in an adequate volume of 10 vol% neutral buffered formalin solution.
STOMACH: The greater curvature of the stomach was incised. The stomach contents were removed by washing with physiological saline. The
stomach piece (included forestomach and glandular stomach) was cut to about 4 × 10 mm size. These parts were fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining part was separated into forestomach part and glandular stomach part. Glandular stomach (two pieces) and forestomach were put into each storage bag and frozen in LN2.
GERM CELLS (Main study): The testes and vas deferens/cauda epididymis were put into a storage bag, separately and squashed then frozen in LN2.
GERM CELLS (Germ cell study): The right and left testis and right and left cauda epididymides were put into a microtube separately and frozen in LN2.
All frozen tissues were stored in an ultra-low temperature freezer.

EXTRACTION OF GENOMIC DNA
- Extraction of genomic DNA in the liver and glandular stomach was conducted.
- In the germ cell, extraction of genomic DNA and examination for mutagenicity was not conducted.
- 3 mL of the buffer for tissue breakage (containing RNase) were poured into a Dounce-type homogenizer and cooled with ice. Each frozen tissue sample was put into the homogeniser and homogenised with a pestle. The homogenised tissue fragments were poured gently into an ice-cooled centrifuge tube containing 3 mL of 0.5 mol/L sucrose solution, and centrifuged at 3000 rotations/min (1710 G) for 10 minutes. The supernatant was removed with a dropper and 3 mL of cooled RNase-containing Dounce buffer were added to the tube and mixed well (nuclear/cell suspension).
- Then, 3 mL of proteinase K solution was added to the nuclear/cell suspension and gently mixed by inversion. This suspension was incubated at 50 °C for about 2 to 2.5 hours until it became clear. The same volume (about 6 mL) of Phenol/Chloroform (Ph/Cl) mixture was added to the solution and mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min (1190 G) for 10 minutes. Next, the upper layer (water layer) was gently collected and transferred into another centrifuge tube by a transfer pipette. This procedure was repeated twice (volume of Ph/Cl mixture was same as the removed water layer). After removal of the water layer, the same volume of chloroform/isoamyl alcohol at a volume ratio of 24:1 was poured into the tube. The contents were mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min (1190 G) for 10 minutes. Then, the water layer was transferred into another centrifuge tube. Genomic DNA was extracted by gradually adding ethanol in the tube. Extracted genomic DNA was transferred into a microtube containing 70 % ethanol and stood for about 10 minutes. The contents were centrifuged at 13,000 rotations/min (13240 G) for 10 minutes. After the supernatant was removed as much as possible using a micropipette, the tube was allowed to stand at room temperature to evaporate ethanol. Appropriate volume (100 μL) of TE buffer was added to the tube. The tube was allowed to stand overnight at room temperature to dissolve DNA residues. The DNA solution was stored in a refrigerator after preparation. The concentration of DNA in the genomic DNA solution was measured using a spectrophotometer and adjusted to about 200 to 600 μg/mL with the TE buffer.

PREPARATION OF TEST STRAINS
-30 mL of LB broth, 300 μL of maltose solution (200 mg/mL), 30 μL of ampicillin solution (50 mg/mL), and 30 μL of kanamycin solution (20 mg/mL) were poured into a 200-mL baffled Erlenmeyer flask. A suspension (50 μL) of Escherichia coli C (lacZ–, gal E–) that had been thawed after being frozen at -80 °C was inoculated into the flask. And it was incubated overnight at 37°C with a shaker at 120 strokes/min as the pre-incubation culture.
- 100 mL of LB broth and 1 mL of maltose solution (200 mg/mL) were poured into a 500-mL baffled Erlenmeyer flask. The pre-incubation culture (1 mL) was inoculated into the flask and it was incubated for about 2 hours (OD: 0.831 to 0.936) under the same conditions as the pre-incubation. Then, the bacterial suspension was centrifuged at 1000 rotations/min for 10 minutes. The supernatant was removed and the cells were suspended in LB broth containing 10 mmol/L magnesium sulfate (E. coli suspension).

PACKAGING OF GENOMIC DNA
- Packaging was conducted according to the instruction manual attached to Transpack packaging extract.
- One red tube of Transpack packaging extract was thawed. Using a pipette, 10 μL of genomic DNA solution was transferred to the red tube. The packaging reaction was mixed by pipetting and the tube was incubated at 30 °C for 90 minutes. Next, a blue tube of Transpack packaging extract was thawed, and 10 μL of it were transferred to the red tube containing a packaging reaction, and mixed in the same manner. It was incubated at 30 °C for another 90 minutes, diluted with 700 μL of SM buffer, and mixed (packaged DNA sample).

PLATING OF PACKAGING DNA
- 1 mL of E. coli suspension for calculating total number of plaques (for tittering) and 2 mL for calculating mutant frequency (for selection) were dispensed into each tube. Then, the entire volume of packaged DNA sample (about 700 μL) was added to E. coli suspension in the tube for selection (total of about 2700 μL) and mixed. The tube was incubated at room temperature for about 30 minutes to allow phage to infect E. coli. 30 μL of the content were diluted 10-fold with 270 μL of LB broth containing 10 mmol/L magnesium sulfate. 30 μL of it were transferred to the tube for tittering and stirred.
The magnesium sulfate solution (1 mol/L) was added to the agar solution at a volume ratio of 2:100 to make the top agar for tittering. The P-gal solution was added to the agar solution at a volume ratio of 2:100 to make the top agar for selection. Then, 17 mL of top agar was added to the tube for tittering and mixed. The contents were poured over a LB agar plate. To the tube for selection, 16 mL of top agar was added and the contents were poured over a LB agar plate in the same manner as the tube for tittering.
- The agar plate was incubated in an incubator at 37 °C overnight.
In the liver of animal ID No. 1105, the above packaging procedure was repeated twice until the total number of plaques per animal reached 300,000.

PLAQUE COUNTING
Calculation of total number of plaques:
- The number of plaques (N) in the plates for tittering was counted, and then the total number of plaques was calculated using the following equation.

Total number of plaques = N × (300 μL/30 μL) × (2 700 μL/30 μL)
= 900 × N

CALCULATION OF NUMBER OF MUTANT PLAQUES
- The number of plaques in the plates for selection was counted and recognized as mutant plaques.

CALCULATION OF MUTANT FREQUENCY
- The lacZ gene was selected as a reporter gene.
- The mutant frequency in a concerned organ was calculated by dividing the number of mutant plaques by the total number of plaques.

Mutant frequency = Number of mutant plaques / Total number of plaques

Evaluation criteria:

The results were evaluated as positive if the mutant frequency for any tissue in any test substance-treated group was significantly different from that in the negative control group. Final judgment includes a comparison against the historical control data and was made in consideration of biological relevance under the test conditions.

Statistics:

The obtained data for in-life phase of this study were collected and analysed using the computer system (LATOX-F/V5).

MUTANT FREQUENCY
- The data on the mutant frequency from the negative control group and each test material-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test material-treated group (two-sided, familywise significance level of 0.05). If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyse the differences.
-The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyse the differences.
The results were evaluated as positive if the mutant frequency for any tissue in any test material-treated group was significantly different from that in the negative control group. Final judgment was include a comparison against the historical control data and was made in consideration of biological relevance under the test conditions.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

LIVER
- In the negative control group, the mean ± SD of mutant frequency among the individuals was 26.9 ± 5.3 (×10^-6).
- The means (±SD) of mutant frequencies in the test material-treated groups, 250, 500 and 1 000 mg/kg/day, were 32.3 ± 8.1 (×10^-6), 30.6 ± 6.6 (×10^-6) and 31.3 ± 3.5 (×10^-6), respectively. All mutant frequencies for the test material-treated groups fell within the laboratory’s acceptable range (based on their historical data) and no statistically significant increase was observed between any of the test group and negative control group.
- The positive control exhibited high and statistically significant mutant frequencies [114.1 ± 14.1 (×10^-6)] compared to those in the negative control (p<0.05).

GLANDULAR STOMACH
- In the negative control group, the mean ± SD of mutant frequency among the individuals was 31.1 ± 9.2 (×10^-6).
- The means (±SD) of mutant frequencies in the test material-treated groups, 250, 500, and 1 000 mg/kg/day, were 30.9 ± 16.2 (×10^-6), 31.6 ± 13.0 (×10^-6) and 32.2 ± 8.6 (×10^-6), respectively. All mutant frequencies for the test material-treated groups fell within the laboratory’s acceptable range (based on their historical data) and no statistically significant increase was observed between any of the test group and negative control group.
- The positive control exhibited high and statistically significant mutant frequencies [287.3 ± 58.9 (×10^-6)] compared to those in the negative control (p<0.05).

BODY WEIGHT AND GENERAL CONDITIONS
- There were no statistically significant differences in body weights between the negative control group and each of the test material-treated groups during the administration period.
- There was no finding in the general condition in any of the test material-treated groups.

FOOD CONSUMPTION
- In the 250, 500 and 1 000 mg/kg/day group, the food consumption from Day 29 to 31 were significantly lower than that in the negative control group. However, these were judged to be not a test material-treatment effect because these were transient and slight changes and not dose-related.

ORGAN WEIGHT AND ABSOLUTE ORGAN WEIGHT / BODY WEIGHT RATIO
- In the 1000 mg/kg/day group, the stomach weight and relative stomach weight to body weight were significantly higher than that in the negative control group.
There were no statistically differences from the negative control group in the liver weight, testes weight or their relative weights to body weight.

GROSS NECROPSY FINDINGS
A cyst in right kidney was observed in the 500 mg/kg/day group. However, this change was considered not to be due to the test material treatment because it was observed in one animal in the middle treatment group only.

Any other information on results incl. tables

Historical Control Data (Transgenic Rodent Gene Mutation Assay (Muta™Mouse:lacZ Assay))

Negative Control

Organ	n	Mutant Frequency [x 10^-6]		Acceptable Range (Mean ± 3 x S.D.)
		Mean ± SD	Min – Max	Lower	Upper
Liver	126	50.1 ± 15.7	19.0 - 95.0	3.0	97.2
Glandular stomach	84	46.2 ± 11.5	24.0 – 84.7	11.7	80.7

 

Positive Control

Organ	n	Mutant Frequency [x 10^-6]
		Mean ± SD	Min – Max
Liver	104	141.8 ± 38.4	84.8 - 285.8
Glandular stomach	59	474.0 ± 123.9	182.4 - 785.3

Negative control: Including water for injection, 0.5 % methylcellulose, corn oil,etc.

Positive control: N-ethyl-N-nitrosourea (ENU, 100 mg/kg)

The above historical control data consists of those pooled from October 26, 2011 to October 13, 2017.

Applicant's summary and conclusion

Conclusions:

Under the conditions in this study the test material did not induce gene mutation in either the liver or glandular stomach of transgenic mice.

Executive summary:

A gene mutation assay with transgenic male mice (Muta™ Mouse) was conducted to assess the potential of the test material to induce gene mutations (reporter gene: lacZ) in the liver and glandular stomach in accordance with the standardised guideline OECD 488.

In a dose-finding study CD2F1/Slc mice were treated for 14 days with 100, 300 and 1 000 mg/kg/day by oral gavage. No deaths or clinical signs of toxicity were observed in any of the dosed groups. Therefore, a limit dosage of 1 000 mg/kg was selected as the high dose and 500 and 250 mg/kg/day were selected as lower doses for the gene mutation assay with Muta™ Mouse.

The test material was administered to male transgenic mice (CD2-LacZ80/HazfBR (Muta™ Mouse)) orally for 28 consecutive days by gavage. In the main study, the liver and glandular stomach were removed after 3 days of manifestation period (Day 29 to 31), and the mutant frequencies were determined. As a result, there were no significant differences in the mutant frequencies in the liver and glandular stomach in any of the groups treated with the test material as compared to the negative control group. The mutant frequencies in the liver and glandular stomach in the positive control group treated with 2 doses of N-ethyl-N-nitrosourea (ENU, dosage level of 100 mg/kg, i.p.) at 24h intervals, were increased. These increases were statistically significant compared with those of the negative control group. No clinical signs, no significant decrease of body weights and no test material-related reduction of food consumption were observed in any groups. There were no test material-related changes noted at necropsy.

In the germ cell study, the testes and cauda epididymis were removed after 49 days of manifestation period (Day 29 to 77). Since no positive response was observed in somatic cells, the mutant frequencies in the germ cells were not evaluated.

It was, therefore, concluded that the test material did not induce gene mutation in the liver or glandular stomach of transgenic mice under the conditions in this study.