Neodymium oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 2007 - 3 August 2007

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:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Government revised Chemical Substance Law according to Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of the Ministry of International Trade and Industry, 9th December 1996.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation 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

Molecular formula:

Nd2O3

IUPAC Name:

dineodymium(3+) trioxidandiide

Test material form:

solid: particulate/powder

Method

Target gene:

S. typhimurium: Histidine locus.
E. coli: Tryptophan locus.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

- 156.3, 312.5, 625, 1250 and 2500 µg/plate, for the TA 102 strain in the first experiment and all tester strains in the second experiment, either with or without S9 mix,
- 312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains except for the TA 102 in the first experiment, either with or without S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in most vehicles used for in vitro tests and so was prepared as an homogeneous suspension.
The test item was suspended at the following concentrations:
100 mg/mL for the preliminary test and the first experiment; 50 mg/mL for the second experiment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes, 37 °C
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATES: three plates/dose-level

POSITIVE CONTROLS
The positive controls were dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water).

Without S9 mix:
-sodium azide (NAN3)
-9-Aminoacridine (9AA)
-2-Nitrofluorene (2NF)
-Mitomycin C (MMC)
-4-Nitroquinoline 1-oxide (4NQO)
With S9 mix:
-2-Anthramine (2AM)
-Benzo(a)pyrene (BaP)

TEST SYSTEM
The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.

Metabolic activation system:
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80 °C, until use.
The S9 mix was prepared at +4 °C immediately before use and maintained at this temperature until added to the overlay agar.

The composition of S9 mix was as follows:
Glucose-6-phosphate 5 mM
NADP 4 mM
KCl 33 mM
MgCl2 8 mM
Sodium phosphate buffer pH 7.4 100 mM
S9 fraction, protein concentration: 36.4 mg/mL 10 % (v/v)
water to volume


EXPERIMENTAL DESIGN
Treatment:
The test material was tested in a preliminary test and two mutagenicity experiments.
The direct plate incorporation method was performed as follows: test material solution (0.05 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45 °C). After rapid homogenisation, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test material solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-response was considered as a positive result. Reference to historical data, or other considerations of biological relevance were taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥156.3 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test material to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 100 µg/plate. At 5000 µg/plate the strong precipitate sometimes (with the TA 102 strain with and without S9 mix) interfered with scoring making the plates unreadable.
No noteworthy toxicity which could be considered as relevant was noted towards the four strains used, with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The control data reported in these report are in the range of the historical control data observed in the laboratory.

Any other information on results incl. tables

Table 2: First experiment (direct plate incorporation) -Mean revertant colony counts

	TA 102			TA 1535			TA 1537
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	314	459	No	11	21	No	8	6	No
156.3	279	413	No (Mp)	-	-	-	-	-	-
312.5	340	478	No (Mp)	11	19	No (Mp)	8	7	No (Mp)
625	292	560	No (Mp)	12	18	No (Mp)	4	7	No (Mp)
1250	234	458	No (Sp)	12	7	No (Mp/Sp)	3	7	No (Sp)
2500	64	302	No (Sp)	6	10	No (Sp)	2	4	No (Sp)
5000	-	-	-	U	5	No (Sp)	U	3	No (Sp)
Positive control	1540	3709	No	491	158	No	448	85	No

	TA 98			TA 100			WP2uvrA
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	20	33	No	151	107	No	27	41	No
312.5	18	28	No (Mp)	150	92	No (Mp)	34	49	No (Mp)
625	18	19	No (Mp)	207	119	No (Sp/Mp)	38	41	No (Mp)
1250	19	22	No (Sp)	215	119	No (Sp/Mp)	30	38	No (Sp)
2500	14	29	No (Sp)	271	109	No (Sp)	16	37	No (Sp)
5000	U	15	No (Sp)	U	88	No (Sp)	U	37	No (Sp)
Positive control	140	1090	No	706	438	No	1347	309	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

U: unreadable

MA : metabolic activation

Table 3: Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) -Mean revertant colony counts

	TA 1535			TA 1537			TA 98
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	15	13	No	4	7	No	30	25	No
156.3	14	13	No (Mp)	8	5	No (Mp)	25	23	No (Mp)
312.5	12	13	No (Mp)	6	9	No (Mp)	22	20	No (Mp)
625	11	10	No (Mp)	7	7	No (Mp)	23	23	No (Mp)
1250	15	9	No (Sp)	3	6	No (Sp)	23	15	No (Sp)
2500	10	4	No (Sp)	2	3	No (Sp)	18	16	No (Sp)
Positive control	577	184	No	351	126	No	192	1144	No

	TA 100			TA 102			WP2 uvrA
Conc. (µg/plate)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	99	138	No	393	520	No	27	24	No
156.3	113	151	No (Mp)	412	426	No (Mp)	29	30	No (Mp)
312.5	125	98	No (Mp)	525	419	No (Mp)	27	24	No (Mp)
625	134	143	No (Mp)	521	581	No (Mp)	28	28	No (Mp)
1250	98	104	No (Sp)	486	562	No (Sp)	27	27	No (Sp)
2500	65	59	No (Sp)	497	658	No (Sp)	25	24	No (Sp)
Positive control	594	493	No	2953	1660	No	806	213	No

*solvent control with DMSO

Mp : Moderate precipitate

Sp : Strong precipitate

U: unreadable

MA : metabolic activation

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative

Under the conditions of this study, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Executive summary:

The potential of the test material to induce reverse gene mutations in Salmonella typhimurium and Escherichia coli was evaluated in a study performed according to the standardised guidelines OECD 471 and EU Method B13/14.

The test material was tested in two independent experiments, with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).

Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 and one strain of Escherichia coli, WP2uvrA, were used. Each strain was exposed to 156.3 to 5000 µg/plate of the test material (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 156.3 µg/plate. No toxicity was noted towards all the strains used either with or without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the six strains.

Under the conditions of the study, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli with and without metabolic activation.