Dipraseodymium trioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2017 to 29 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration in peripheral human lymphocytes

Test material

Test material information

Constituent 1

Reference substance name:

Dipraseodymium trioxide

EC Number:

234-845-3

EC Name:

Dipraseodymium trioxide

Cas Number:

12036-32-7

Molecular formula:

O3Pr2

IUPAC Name:

Praseodymium (III) oxide

Test material form:

solid

Details on test material:

- Appearance: Pale green

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: PR2O3-4/16
- Expiration date of the lot/batch: 21 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark over silica gel, the test material was held under argon gas for shipment and batched into individual containers under Nitrogen for use in the study to avoid exposure to the atmosphere. A new sample was used for each experiment.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Sex, age and number of blood donors if applicable: males aged 23 and 24
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The molecular weight of the test material was given as 329.81, therefore, the maximum dose level was 2000 µg/mL, the maximum recommended dose level.

- Preliminary toxicity test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
- Main experiment: 0.125, 0.25, 0.5, 1, 2, 4 and 8 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM)
- Justification for choice of solvent/vehicle:The test material was insoluble in both dimethyl sulphoxide and acetone at 200 mg/mL but was partially soluble/ suspendable in Minimal Essential Medium (MEM) at 20 mg/mL in solubility checks performed in house.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

EXPOSURE GROUPS

4-HOUR EXPOSURE WITH METABOLIC ACTIVATION (S9)
After approximately 48 hours incubation at approximately 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37°C, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37°C in 5% CO2 in humidified air.

4-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION
After approximately 48 hours incubation at approximately 37°C with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test material solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37°C, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION
As the exposure was continuous the cultures were established, at a nominal volume of 9.0 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test material or 0.1 mL of positive control. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37°C, 5% CO2 in humidified air for 24 hours.

PRELIMINARY TOXICITY TEST
- The preliminary toxicity test was performed using all three of the exposure conditions as described above but using single cultures only.
- The dose range of the test material used was 7.81 to 2000 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
- Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main test.

MAIN EXPERIMENT
The three exposure groups above were used for the main study with the following test material concentrations: 0.125, 0.25, 0.5, 1, 2, 4 and 8 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

CELL HARVEST
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4°C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS AND STAINING
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
- When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

EVALUATION OF RESPONSE
- Qualitative Slide Assessment: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.
- Coding: The slides were coded using a computerized random number generator.
- Mitotic Index: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Scoring of Chromosome Damage: Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of endoreduplicated cells) was also reported.

Evaluation criteria:

DATA EVALUATION
The following criteria were used to determine a valid assay:
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory control data range.
- All the positive control chemicals induced a positive response (p ≤ 0.01) demonstrating the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analysed.

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
A test material can be considered to be clearly negative if, in any of the experimental conditions examined:
- The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
- No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
- There is no concentration-related increase at any dose level.

A test material can be classified as genotoxic if:
- The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
- At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
- The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.

When all of the above criteria are met, the test material can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
- A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 125 µg/mL, in the absence of S9 and at and above 62.5 µg/mL in the presence of S9. Precipitate was observed in the blood pellet of the cultures at the end of the exposure period at all test material dose levels in all three exposure groups.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2000 µg/mL in all three exposure groups. Precipitate from the test material was noted on the slides at all test material dose levels and was particularly heavy at and above 500 µg/mL in all three exposure groups. The test material induced no evidence of toxicity in any of the exposure groups.
- The selection of the maximum dose level for the Main Experiment was based on the lowest dose level where precipitate was observed and was 8 µg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.

MAIN EXPERIMENT
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 8 µg/mL in all three exposure groups.
- Precipitate observations were made at the end of exposure in the blood-free cultures and precipitate was noted at 8 µg/mL in the presence of S9 only. Precipitate was observed in the cell pellet of the blood cultures at the end of exposure at and above 0.5 µg/mL in the 4(20)-hour exposure groups, and at and above 2 µg/mL in the 24-hour continuous exposure group. These were considered to be the lowest precipitating dose levels.
- The mitotic index data for the Main Experiment confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the three exposure groups.
- The maximum dose level selected for metaphase analysis was the lowest precipitating dose level seen in the blood cultures at the end of exposure and was 0.5 µg/mL in the 4(20)-hour exposure groups and 2 µg/mL for the 24-hour exposure group.
- The mitotic index and chromosome aberration data are given in Table 1, Table 2 and Table 3.

- The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

Any other information on results incl. tables

Table 1: Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of Aberrations						Total number of aberrations		Frequency of aberrant cells (%)
					Chromatid		Chromosome		Others	(+ Gaps)	(- Gaps)	(+ Gaps)	(-Gaps)
				Gaps	Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	4.40	150	0	0	0	0	0	0	0	0	0	0
	B	4.60	150	0	1	0	0	0	0	1	1	1	1
	Total	(100)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
0.125µg/mL	A	5.30	150	0	0	0	0	0	0	0	0	0	0
	B	4.50	150	0	0	0	0	0	0	0	0	0	0
	Total	(109)	300	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)
0.25 µg/mL	A	2.85	150	0	0	0	0	0	0	0	0	0	0
	B	6.60	150	0	0	0	0	0	0	0	0	0	0
	Total	(105)	300	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)
0.5 µg/mL	A	3.25	150	0	0	0	0	0	0	0	0	0	0
	B	5.45	150	0	1	0	0	0	0	1	1	1	1
	Total	(97)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
Positive control (MMC 0.4 µg/mL)	A	4.10	150	0	6	4	1	0	0	11	11	9	9
	B	1.35	83*	1	15	4	1	0	0	21	20	15	15
	Total	(61)	233	1	21	8	2	0	0	32	31	24 (10.3)	24** (15.2)

MMC = Mitomycin C

* = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

** = P < 0.001

MEM = Minimal Essential Medium

Table 2: Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (2% S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of Aberrations						Total number of aberrations		Frequency of aberrant cells (%)
					Chromatid		Chromosome		Others	(+ Gaps)	(- Gaps)	(+ Gaps)	(-Gaps)
				Gaps	Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	6.45	150	0	0	0	0	0	0	0	0	0	0
	B	3.45	150	0	1	0	0	0	0	1	1	1	1
	Total	(100)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
0.125µg/mL	A	6.95	150	0	3	0	1	0	0	4	4	3	3
	B	3.40	150	0	0	0	1	0	0	1	1	1	1
	Total	(105)	300	0	3	0	2	0	0	5	5	4 (1.3)	4 (1.3)
0.25 µg/mL	A	5.45	150	0	0	0	1	0	0	1	1	1	1
	B	5.80	150	0	0	0	1	0	0	1	1	1	1
	Total	(114)	300	0	0	0	2	0	0	2	2	2 (0.7)	2 (0.7)
0.5 µg/mL	A	4.75	150	0	0	0	0	0	0	0	0	0	0
	B	4.30	150	1	0	0	0	0	0	1	0	1	0
	Total	(91)	300	1	0	0	0	0	0	1	0	1 (0.3)	0 (0.0)
Positive control (CP 2 µg/mL)	A	2.85	129*	1	14	2	1	0	0	18	17	16	15
	B	3.25	135*	4	10	4	4	0	0	22	18	18	15
	Total	(62)	264	5	24	6	5	0	0	40	35	34 (12.9)	30** (11.4)

 

CP = cyclophosphamide

* = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

** = P < 0.001

MEM = Minimal Essential Medium

Table 3: Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure Without Metabolic Activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of Aberrations						Total number of aberrations		Frequency of aberrant cells (%)
					Chromatid		Chromosome		Others	(+ Gaps)	(- Gaps)	(+ Gaps)	(-Gaps)
				Gaps	Breaks	Exchanges	Breaks	Exchanges	X
Vehicle control (MEM)	A	9.30	150	1	2	0	0	0	0	3	2	2	2
	B	11.30	150	0	0	0	0	0	0	0	0	0	0
	Total	(100)	300	1	2	0	0	0	0	3	2	2 (0.7)	2 (0.7)
0.25µg/mL	A	7.55	150	0	1	0	0	0	0	1	1	1	1
	B	9.30	150	0	0	0	0	0	0	0	0	0	0
	Total	(82)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
0.5 µg/mL	A	7.45	150	0	0	0	0	0	0	0	0	0	0
	B	7.60	150	0	1	0	0	0	0	1	1	1	1
	Total	(73)	300	0	1	0	0	0	0	1	1	1 (0.3)	1 (0.3)
1 µg/mL	A	6.15	150	1	1	0	1	0	0	3	2	3	2
	B	9.40	150	1	0	0	0	0	0	1	0	1	0
	Total	(75)	300	2	1	0	1	0	0	4	2	4 (1.3)	2 (0.7)
2 µg/mL	A	9.35	150	1	0	0	0	0	0	1	0	1	0
	B	6.50	150	0	0	0	0	0	0	0	0	0	0
	Total	(77)	300	1	0	0	0	0	0	1	0	1 (0.3)	0 (0.0)
Positive control (MMC 0.2 µg/mL)	A	2.90	31*	2	19	6	1	0	0	28	26	18	18
	B	3.65	43*	0	6	7	3	0	0	16	16	15	15
	Total	(32)	74	2	25	13	4	0	0	44	42	33 (44.6)	33** (44.6)

MMC = mitomycin C

* = Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

** = P < 0.001

MEM = Minimal Essential Medium

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test material was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The potential of the test material to cause chromosomal aberrations in human lymphocytes was determined in accordance with the standardised guidelines OECD 473 and The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE), under GLP conditions.

During the study, duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were:0.125, 0.25, 0.5, 1, 2, 4, 8 µg/mL.

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive controls induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. 

Under the conditions of this study, the test material was considered to be non-clastogenic to human lymphocytes in vitro.