reaction mass of neodymium carbonate and praseodymium carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-APR-2013 to 16-DEC-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in compliance with GLP and according to the OECD guideline 473.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

cited as Directive 84/449/EEC, B.10

Deviations:

yes

Remarks:

(see below)

Principles of method if other than guideline:

Deviations:
Due to a technical error, only one culture was available for the dose-level of 1250 μg/mL in the second experiment with S9 mix.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of neodymium carbonate and praseodymium carbonate

Molecular formula:

(Nd,Pr)2 (CO3)3

IUPAC Name:

reaction mass of neodymium carbonate and praseodymium carbonate

Constituent 2

Reference substance name:

937-221-5

IUPAC Name:

937-221-5

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

Species / strain / cell type:

primary culture, other: human lymphocytes

Details on mammalian cell type (if applicable):

Cultures of human lymphocytes were prepared from whole blood samples obtained from healthy, non-smoking donors and collected into heparinized sterile tubes.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (Moltox, Molecular Toxicology, INC, Boone, USA), obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route, was used as the metabolic activation system.

Test concentrations with justification for top dose:

Nominal concentrations corrected to take into account the water content (i.e. correction factor = 1.58)
Experiment 1: 0, 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500, and 5000 µg/mL with or without metabolic activation
Experiment 2: 0, 78.13, 156.3, 312.5, 625, 1250, and 2500 µg/mL with or without metabolic activation

Vehicle / solvent:

- Vehicle used: Water (batch Nos. 2F2843, 3F0043, 3F0282)
- Justification for choice of solvent/vehicle: A homogenous suspension of the test item was obtained at the concentration of 100 mg/mL in water (after 20-min ultrasonication).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(MMC) without metabolic activation: at 2 and 3 µg/mL for the 3-h treatment and at 0.3 and 0.5 µg/mL for the continuous treatment

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(CPA) with metabolic activation: at 12.5 and 25 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In suspension

DURATION (see table 1 below for details)
- Exposure duration: 3, 20, or 44 h
- Expression time: 0, 14, or 38 h
- "Selection" time: 3 h before harvesting
- Fixation time: 20, or 44 h

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 1000 cells per culture for cytotoxicity evaluation; 200 metaphases/dose-level for cytogenetic analysis

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index [MI] (number of cells in mitosis/number of cells examined)

OTHER:
- Scoring method: The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy, endoreduplication and hyperdiploidy.

Evaluation criteria:

ACCEPTANCE CRITERIA
The study was considered valid if all the following criteria were met:
- the frequency of cells with structural chromosome aberration in the vehicle controls was consistent with (but not necessary within) the historical data. In any case, this frequency should be ≤ 5%,
- the frequency of cells with structural chromosome aberration in the positive controls was statistically higher than that of the vehicle controls (p ≤ 0.05) and consistent with (but not necessary within) the historical data.

EVALUATION CRITERIA
- Evaluation of a positive response: a test item was considered positive for inducing chromosomal aberrations if a reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration was observed at one or more dose levels and at one or two harvest times.
- Evaluation of a negative response: a test item was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations for any of the dose levels and at any harvest times.

Statistics:

For each experiment and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the χ² test unless treated culture data were lower than or equal to the vehicle control data. P = 0.05 was be used as the lowest level of significance.

Results and discussion

Test results

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see in "any other information on results incl. tables" below for details

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see in "any other information on results incl. tables" below for details

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

- DETAILED RESULTS:

 

Table 1: Cytotoxicity data

Doses^(1) µg/mL	Mean MI as % of the control				Decrease in MI (%)
	- S9		+ S9		- S9		+ S9
Experiment 1 0	3-h expo/17-h recov 100		3-h expo/17-h recov 100		3-h expo/17-h recov -		3-h expo/17-h recov -
39.06	102		88		none		12
78.13	94		82		6		18
156.3	107		87		none		13
312.5	98		88		2		12
625	73		88		27		12
1250	74		76		26		24
2500 P	-		-		-		-
5000 P	-		73		-		27
MMC 2 µg/mL	35				65
MMC 3 µg/mL	19				81
CPA 12.5 µg/mL			34				66
CPA 25 µg/mL			21				79
Experiment 2     0	20-h expo     100	44-h expo     100	3-h expo/ 17-h recov 100	3-h expo/ 41-h recov 100	20-h expo     -	44-h expo     -	3-h expo/ 17-h recov -	3-h expo/ 41-h recov -
78.13	88	120	87	129	12	none	13	none
156.3	89	105	78	109	11	none	22	none
312.5	61	107	66	101	39	none	34	none
625 P	80	86	63	80	20	14	37	20
1250 P	86	-	89	-	14	-	11	-
2500 P	59	-	76	-	41	-	24
MMC 0.3 µg/mL	29				71
MMC 0.5 µg/mL	31				69
CPA 12.5 µg/mL			28				72
CPA 25 µg/mL			30				70

⁽¹⁾: expressed as active item

CPA: cyclophosphamide

expo: exposure

MI: mitotic index

MMC: mitomycin C

P: precipitate observed in the culture medium at the end of treatment

recov: recovery

 

Table 2: Genotoxicity data

Doses^(1) µg/mL	Cells with structural chromosome aberrations
	Frequency (%) including gaps				Frequency (%) excluding gaps
	- S9		+ S9		- S9		+ S9
Experiment 1 0	3-h expo/17-h recov 0.0		3-h expo/17-h recov 0.0		3-h expo/17-h recov 0.0		3-h expo/17-h recov 0.0
39.06
78.13
156.3
312.5	0.0		0.0		0.0		0.0
625	1.5		0.5		1.0		0.0
1250	0.5		0.0		0.5		0.0
2500 P
5000 P
MMC 2 µg/mL
MMC 3 µg/mL	41.0				40.0***
CPA 12.5 µg/mL			22.0				21.0***
CPA 25 µg/mL
Experiment 2     0	20-h expo     0.5	44-h expo     1.0	3-h expo/ 17-h recov 0.0	3-h expo/ 41-h recov 1.5	20-h expo     0.5	44-h expo     1.0	3-h expo/ 17-h recov 0.0	3-h expo/ 41-h recov 1.0
78.13
156.3	1.0		2.0		1.0		2.0
312.5	1.0		2.5		1.0		2.5
625 P	2.5	2.5	3.0	0.0	1.5	2.5	3.0*	0.0
1250 P
2500 P
MMC 0.3 µg/mL	23.0				23.0***
MMC 0.5 µg/mL
CPA 12.5 µg/mL			18.0				17.0***
CPA 25 µg/mL

⁽¹⁾: expressed as active item

CPA: cyclophosphamide

expo: exposure

MI: mitotic index

MMC: mitomycin C

P: precipitate observed in the culture medium at the end of treatment

recov: recovery

Statistical analysis: χ² test 

***: p < 0.001 (performed only for cells with structural aberrations excluding gaps)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions of this study, the test item, Reaction mass of neodymium carbonate and praseodymium carbonate, did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, reaction mass of neodymium carbonate and praseodymium carbonate, to induce chromosome aberrations in cultured human lymphocytes according to the OECD guideline 473 and in compliance with GLP.

 

The test item, suspended in water for injections, was tested at concentrations up to 5000 µg/mL (act. ingr.) in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. Lymphocyte cultures were exposed to the test or control items for 3 hours (with S9 mix) and 3, 20, or 44 hours (without S9 mix) then rinsed. Following exposure to the test or control items with S9 mix, the cultures were incubated in fresh medium at +37°C until harvest. Harvest times were 20 and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

The cytotoxicity was indicated by the reduction of mitotic index (MI). Three hours before harvest, each culture was treated with a Colcemid® solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

 

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered to be valid.

 

Regarding the first experiment, the dose-levels selected for the treatment were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL, both with and without S9 mix. A precipitate was observed in the culture medium at the end of the treatment period, at dose levels ≥ 2500 µg/mL. Moreover, following the 3-hour treatment and the 20-hour harvest time, a slight cytotoxicity was observed at dose-levels ≥ 625 µg/mL without S9 mix (26-27%) and at 5000 µg/mL with S9 mix (27%). However, no significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment with or without S9 mix.

 

In the second experiment, the dose-levels selected were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/mL both with and without S9 mix. A precipitate was observed in the culture medium at the end of the treatment periods, at dose levels ≥ 625 µg/mL. Further, a moderate cytotoxicity was noted at 312.5 and 2500 µg/mL (39 and 41%, respectively) following the 20-hour treatment without S9 mix. In the presence of S9 mix, a slight cytotoxicity was noted at 312.5 and 625 µg/mL (34 and 37%, respectively), at the 20-hour harvest time only. Nevertheless, no significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-, 20- and 44-hour treatments, except for a statistically significant increase at 625 µg/mL (3.0% vs. 0.0% for the vehicle control) following the 20-hour treatment with S9 mix. However, this increase remained within the vehicle control historical range (0.0 to 4.5%) and no similar effect was observed in both experiments. Thus the slight increase observed in the second experiment was not considered to be biologically relevant.

 

In conclusion, these results were considered to meet the criteria of a negative response in both experiments. Consequently, under the experimental conditions of this study, the test item, reaction mass of neodymium carbonate and praseodymium carbonate, did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a rat liver metabolizing system.