Neodymium trifluoride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2012 and 25 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Neodymium trifluoride

EC Number:

237-253-3

EC Name:

Neodymium trifluoride

Cas Number:

13709-42-7

Molecular formula:

F3Nd

IUPAC Name:

neodymium trifluoride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: Pale brown powder
Storage Conditions: Room temperature in the dark

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Strain: CHO-K1
- Type and identity of media: Cells were routinely cultured in Ham's F12 medium supplemented with 5 % foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 µg/mL) at 37 °C with 5 % CO₂ in air.
- Properly maintained: yes. Stocks of cells were maintained in liquid nitrogen at approximately -196 °C.
- Periodically "cleansed" against high spontaneous background: yes.Before the stokes of cells were frozen, they were cleansed of spontaneous mutants by culturing HAT medium (Ham's F12 medium supplemented with Hypoxanthine 13.6 µg/mL, 100 µM; Aminopterin (0.0178 µg/mL, 0.4 µm; and Thymidine 3.85 µg/mL, 16 µm) for four days. The cells were then passaged into HAT free medium and cultured for 4 to 7 days before being frozen.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (10 and 20 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 (4 hour incubation period):
Without metabolic activation: 0, 62.88, 125.75, 251.5, 503, 1006 and 2012 µg/mL
With metabolic activation: 0, 62.88, 125.75, 251.5, 503, 1006 and 2012 µg/mL

Experiment 2 (24 hour incubation period):
Without metabolic activation: 0, 31.44, 62.88, 125.75, 251.5, 503, 1006 and 2012 µg/mL
With metabolic activation: 0, 62.88, 125.75, 251.5, 503, 1006 and 2012 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none (the test material was accurately weighed and suspended directly in Ham's F12 culture medium)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. For the 4 hour exposure groups of Experiment 1 and Experiment 2, cells were seeded at 1.5 x 10⁶/75 cm² flask approximately 48 hours before being exposed to the test or control materials. For the 24 hour exposure groups of Experiment 2, cells were seeded at 1.0 x 10⁶/75 cm² flask approximately 48 hours before being exposed to the test or control materials. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test material, vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham’s F12) at 37 °C in an incubator with a humidified atmosphere of 5 % CO₂ in air.

At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Ham’s F12 with 5 % FBS. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10⁶ cells/flask in a 225 cm² flask to allow growth and expression of induced mutants, and in triplicate in 25 cm² flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in Ham’s F12 with 5 % FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5 % CO₂ in air.

Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10⁶ cells/225 cm² flask on day 3 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:

i) In triplicate at 200 cells/25 cm² flask in 5 mL of Ham’s F12 with 5 % FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10⁵ cells/75 cm² flask (5 replicates per group) in Ham’s F12 with 5 % FBS, supplemented with 10 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37 °C in an incubator with humidified atmosphere of 5 % CO₂ in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage of viability and mutation frequency per survivor were calculated for each dose group.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10 % Giemsa solution for 5 minutes.


DURATION
- Exposure duration: 4 hours - Experiment 1; 24 hours - Experiment 2
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): 10 µg/mL 6-Thioguanine (6-TG)
STAIN : Giemsa

NUMBER OF REPLICATIONS: Test concentrations were performed in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency and reduction in mutant frequency.

Evaluation criteria:

EVALUATION OF RESULTS
The test material was considered positive if an increase in mutant frequency per survivor which exceeded the negative control by 20 x 10⁻⁶ mutants per viable cell.

CLONING EFFICIENCY
The cloning efficient (CE) and % control was calculated using the following equations:
CE % = (mean CE counts/200) x 100

% Control = (CE% of Dose IDx/CE % of Dose ID₀) x 100

MUTANT FREQUENCY
Mutant frequency/10⁶ and mutant frequency survival rate (MFSV) were calculated using the following:
MF 10⁻⁶ for each dose = Total mutant plate counts
MFSV for each dose = (MF⁻⁶/CE) x 100
Dose ID₅₀ = Vehicle control values
Dose IDx = Dose level values

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The 4 hour group with and without metabolic activation demonstrated a reduction in cloning efficiency of 41 % and 29 %, respectively. The 24 hour group gave a 47 and 84 % reduction in cloning efficiency at 125.75 and 251.5 µg/mL respectively.

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment 1, a precipitate of the test material was seen at the end of exposure at and above 251.5 µg/mL in the absence and presence of metabolic activation. In experiment 2, a precipitate of the test material was observed at the end of the exposure period at all concentrations in the presence of metabolic activation, and at and above 125.75 µg/mL in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A dose range of 7.86, 15.72, 31.44, 62.88, 125.75, 251.5, 503, 1006 and 2012 µg/mL was used in a preliminary cytotoxicity test. 2012 µg/mL is the maxmimum recommended concentration equivalent to 10 mM. A precipitate was observed at the end of the exposure at and above 62.88 µg/mL in all exposure groups. There was a reduction in cloning efficiency in the upper dose range in all three exposure groups. The toxicity was particularly marked in the 24 hour exposure group. The maximum dose level for the mutagenicity test was selected on the basis of these findings.

COMPARISON WITH HISTORICAL CONTROL DATA: The data was in agreement with historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In experiment 1, the cloning efficiencies for the 4 hour exposure group in the presence of metabolic activation did not achieve 70 %, however it was greater than 50 % and was therefore considered acceptable.
In experiment 2, there was a modest reduction in cloning efficiency in the 4 hour exposure group of 24 % at the maximum dose tested. In the 24 hour exposure group, the 503 and 1006 µg/mL concentrations did not reach greater than 10 % cloning efficiency, but were plated as part of the toxicity plateau. Furthermore, the cloning efficiencies for day 7 vehicle control, for the 24 hour exposure group, and in the day 0 vehicle control cloning efficiencies for the 4 hour exposure group, in the presence of metabolic activation did not achieve 70 %, however it was greater than 50 % and was therefore considered acceptable.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Experiment 1

Exposure Time (hours)	Dose (µg/mL)	Replicate	Day 7 Mutant
			MF	MFS 10⁻⁶	SD	Group MFS 10^-6
4 (-S9)	0	A	14	13.7	0.92	17
		B	18	20.1
	62.88	A	13	13.1	0.82	16
		B	17	18.6
	125.75	A	10	10.7	1.25	10
		B	7	8.9
	251.5*	A	10	11.6	1.63	20
		B	20	28.4
	503*	A	26	29.8	2.42	25
		B	9	20.6
	1006*	A	16	16.8	1.52	19
		B	15	22.1
	2012*	A	27	29.5	1.58	29
		B	18	27.8
	EMS 500	A	106	130.1	5.25	133
		B	87	135.9
	EMS 750	A	99	139.4	5.51	158
		B	132	176.0
4 (+S9)	0	A	22	21.1	1.35	22
		B	23	22.9
	62.88	A	10	10.6	1.43	12
		B	14	12.4
	125.75	A	10	10.6	1.83	17
		B	23	24.1
	251.5*	A	9	9.1	1.66	15
		B	20	21.9
	503*	A	17	18.0	1.93	24
		B	31	29.5
	1006*	A	23	24.0	1.51	29
		B	32	33.0
	2012*	A	24	23.0	1.26	21
		B	20	20.0
	DMBA 0.5	A	118	171.4	10.84	223
		B	192	274.3
	DMBA 1	A	158	300.0	5.39	274
		B	114	247.8

 

* precipitate

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

EMS = Ethyl methane sulphonate

DMBA = Dimethyl benzanthracene

 

Table 2 Experiment 2

Exposure Time (hours)	Dose (µg/mL)	Replicate	Day 7 Mutant
			MF	MFS 10⁻⁶	SD	Group MFS 10^-6
24 (-S9)	0	A	13	13.9	1.37	21
		B	18	27.1
	31.44	A	7	8.6	1.75	21
		B	21	33.2
	62.88	A	8	9.3	1.05	14
		B	12	18.2
	125.75*	A	13	14.7	1.48	19
		B	15	23.1
	251.5*	A	6	8.5	2.12	18
		B	19	27.7
	503*	A	10	14.1	0.95	12
		B	7	10.0
	1006*	A	18	25.1	0.79	30
		B	20	35.2
	2012*	A	7	10.5	1.32	12
		B	15	13.7
	EMS 200	A	179	271.2	9.27	255
		B	252	239.6
	EMS 300	A	209	435.4	6.96	415
		B	227	394.8
4 (+S9)	0	A	17	16.1	1.16	19
		B	20	21.1
	62.88*	A	24	23.0	1.43	22
		B	21	21.2
	125.75*	A	19	19.3	1.20	16
		B	12	13.2
	251.5*	A	19	18.9	1.41	23
		B	21	27.2
	503*	A	21	22.4	1.55	21
		B	17	19.0
	1006*	A	27	30.9	1.73	29
		B	24	26.2
	2012*	A	31	31.0	2.91	27
		B	22	22.4
	DMBA 0.5	A	272	463.6	6.47	451
		B	224	439.2
	DMBA 1	A	220	474.8	9.94	529
		B	252	583.8

 

* precipitate

MF = Mutant frequency

MFS = Mutant frequency per survivor

EMS = Ethyl methane sulphonate

DMBA = Dimethyl benzanthracene

 

 

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.

Executive summary:

A study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells in vitro in accordance with the standardised guidelines OECD 476, EU Method B.17, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the EPA OPPTS 870.5300.

CHO cells were treated with the test material at six dose levels, in duplicate, together with negative and positive controls. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.

Two treatment conditions were used for the test. In Experiment 1, a 4 hour exposure in the presence of 2 % S9 and in the absence of metabolic activation. In Experiment 2, the 4 hour exposure was repeated using a 1 % final S9 concentration, whilst in the absence of metabolic activation the 4 -hour exposure was repeated with to verify a possible response seen in Experiment 1.

The negative (Ham’s F12 culture medium) controls gave mutant frequencies within the range expected for CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

 

The test material did not induce any significant or dose-related increases in mutant frequency per survivor at any dose level in the presence of metabolic activation in either of the two experiments.

Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.