Neodymium trihydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 August 2016 to 9 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

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 trihydroxide

EC Number:

240-514-4

EC Name:

Neodymium trihydroxide

Cas Number:

16469-17-3

Molecular formula:

H3NdO3

IUPAC Name:

neodymium trihydroxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): neodymium trihydroxide
- Physical state: solid
- Appearance: clear blue powder (purple under UV light)

Specific details on test material used for the study:

- correction factor: correction factor of 1.17 was used at dose formulation preparation

Method

Target gene:

Histidine locus / tryptophan locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: excision repair defects or muc+ gene

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial S9 fraction (rat liver)

Test concentrations with justification for top dose:

5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate in the absence and presence of metabolic activation. The choice of the concentrations was done on the basis of a Preliminary Compatibility Test and a Preliminary Concentration Range Finding Test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). The formulations at 100 mg/mL concentration using DMSO or DMF as vehicles were suspensions with fast sedimentation. However, the formulation at the same concentration using distilled water was a suspension with slow sedimentation and was suitable for the test (with continuous stirring of the formulations).
- Preparation: Distilled water was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

INITIAL MUTATION TEST
- Method of application: in agar (plate incorporation)
- Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and in absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes: top agar 2000 µL; vehicle or test item formulation (or reference controls) 50 µL; overnight culture of test strain: 100 µL; phosphate buffer (pH 7.4) or S9 mix 500 µL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48+/-1 hours.

CONFIRMATION MUTATION TEST (pre-incubation method)
- A pre-incubation procedure was performed as a Confirmatory Mutation test once no positive effect was observed in the Initial Mutation Test.
- Bacteria (cultured in Nutrient Broth no. 2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
- Before overlaying, the test item formulation (or vehicle/solvent or reference control) , the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37°C in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48+/-1 hours.

EVALUATION OF EXPERIMENTAL DATA
- The colony numbers of the untreated/negative (solvent)/positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or sign of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM Software. Mutation Factor = mean number of revertants on the test item plate/mean number of revertants on the vehicle control plate

In the main tests, each sample (including the controls) was tested in triplicate.

Evaluation criteria:

A test item was considered mutagenic if:
- A concentration-related increase in the number of revertants occurs and/or;
- A reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- The number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and E. coli WP2 uvrA bacterial strains;
- The number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a negative response:
A test article was considered non-mutagenic if: the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or E. coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control. The negative response should be reproducible in at least one follow up experiment.

Statistics:

Manual colony counts were performed on the plates. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Concentration Range Finding Test:
- The observed numbers of revertant colonies was in the normal range. Slight decreases of the revertant counts were observed compared to the solvent control sporadically. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
- Precipitate/slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000, 2500 and 1000 µg/plate. This made the evaluation of the background lawn development difficult at 5000 µg and 2500 µg/plate concentrations.
- Inhibitory or toxic effects of the test item were not detected in the Preliminary Range Finding Test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the Inital Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain with metabolic activation at the concentration of 5 µg/plate. The mutation factor value was 1.61. However, there was no dose-resonse relationship, the observed mutation factor values were below the biologically relevant treshold limit and the number of revertant colonies was within the historical control range.
In the Confirmation Mutation Test (pre)incubation method), the highest revertant rate was observed in E. coli WP2 uvrA bacterial strain at 158.1 µg/plate concentration with metabolic activation. THe calculated mutation factor value at this dose level was 1.43. However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Slight increases in the numbers of revertant colonies were detected compared to the solvent control during the study in some cases. However, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and were within the historical control range. They were considered as reflecting the biological variability of the test.

Any other information on results incl. tables

Validity of the tests

Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the amin tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range-finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Summary table of the initial mutation test:

Concentrations (µg/plate)	mean values of revertants/mutation factor (MF)	Salmonella typhimurium tester strains								Eschericha coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
untreated control	mean	23.3	28.0	122.7	122.7	14.0	9.3	9.7	13.3	38.3	33.7
	MF	0.90	1.09	1.18	0.97	0.91	0.90	1.04	1.11	1.12	0.75
DMSO control	mean	31.7	33.3	-	131.7	-	11.0	10.7	14.0	-	34.7
	MF	1.22	1.30	-	1.04	-	1.06	1.14	1.17	-	0.78
distilled water control	mean	26.0	25.7	104.0	126.3	15.3	10.3	9.3	12.0	34.3	44.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	mean	27.3	28.7	84.0	98.0	10.7	9.3	11.7	11.0	28.7	29.0
	MF	1.05	1.12	0.81	0.78	0.70	0.90	1.25	0.92	0.83	0.65
1581	mean	30.7	30.0	94.3	100.3	12.3	12.0	9.7	15.0	32.0	35.0
	MF	1.18	1.17	0.91	0.79	0.8	1.16	1.04	1.25	0.93	0.78
500	mean	25.7	29.0	95.0	118.3	12.3	12.3	10.3	10.7	35.3	39.3
	MF	0.99	1.13	0.91	0.94	0.80	1.19	1.11	0.89	1.03	0.88
158.1	mean	26.3	30.0	92.3	120.7	12.3	9.0	11.3	14.3	34.0	36.7
	MF	1.01	1.17	0.89	0.96	0.80	0.87	1.21	1.19	0.99	0.82
50	mean	30.0	28.3	110.0	120.3	12.7	16.3	14.7	12.3	33.3	34.3
	MF	1.15	1.10	1.06	0.95	0.83	1.58	1.57	1.03	0.97	0.77
15.81	mean	21.3	32.7	111.0	136.0	13.7	10.0	13.3	15.3	37.0	38.7
	MF	0.82	1.27	1.07	1.08	0.89	0.97	1.43	1.28	1.08	0.87
5	mean	26.3	29.0	107.0	121.7	16.0	8.7	7.7	19.3	37.0	35.7
	MF	1.01	1.13	1.03	0.96	1.04	0.84	0.82	1.61	1.08	0.80
NPD 4 µg	mean	343.7	-	-	-	-	-	-	-	-	-
	MF	10.85	-	-	-	-	-	-	-	-	-
2AA 2 µg	mean	-	2488.0	-	2481.3	-	213.0	-	212.7	-	-
	MF	-	74.64	-	18.85	-	19.36	-	15.19	-	-
2AA 50 µg	mean	-	-	-	-	-	-	-	-	-	204.7
	MF	-	-	-	-	-	-	-	-	-	5.90
SAZ 2 µg	mean	-	-	1042.7	-	1032.0	-	-	-	-	-
	MF	-	-	10.03	-	67.30	-	-	-	-	-
9AA 50 µg	mean	-	-	-	-	-	-	398.0	-	-	-
	MF	-	-	-	-	-	-	37.31	-	-	-
MMS 2 µL	mean	-	-	-	-	-	-	-	-	936.0	-
	MF	-	-	-	-	-	-	-	-	27.26	-

Summary table of confirmatory mutation test:

Concentration (µg/plate)	mean values of revertants/mutation factor	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	mean	19.7	36.7	113.3	123.0	14.7	14.7	7.0	8.7	40.7	29.0
	MF	0.88	0.98	1.05	1.09	1.05	1.19	0.70	0.87	0.89	0.89
DSMO control	mean	22.7	31.0	-	110.0	-	11.3	11.3	8.0	-	32.3
	MF	1.01	0.83	-	0.98	-	0.92	1.13	0.80	-	0.99
Distilled water control	mean	22.3	37.3	107.7	112.3	14.0	12.3	10.0	10.0	45.7	32.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	mean	21.0	28.0	80.0	75.0	9.3	11.0	4.3	6.0	34.3	44.7
	MF	0.94	0.75	0.74	0.67	0.67	0.89	0.43	0.60	0.75	1.37
1581	mean	25.7	33.0	107.7	123.7	12.3	10.0	7.3	9.0	39.3	44.0
	MF	1.15	0.88	1.00	1.10	0.88	0.81	0.73	0.90	0.86	1.35
500	mean	28.0	26.7	125.7	135.7	14.0	12.0	8.7	6.3	42.0	46.3
	MF	1.25	0.71	1.17	1.21	1.00	0.97	0.87	0.63	0.92	1.42
158.1	mean	28.7	31.7	124.3	130.3	14.7	11.7	8.3	9.3	43.0	46.7
	MF	1.28	0.85	1.15	1.16	1.05	0.95	0.83	0.93	0.94	1.43
50	mean	25.0	32.3	109.3	128.3	13.0	13.3	8.7	6.3	42.3	41.7
	MF	1.12	0.87	1.02	1.14	0.93	1.08	0.87	0.63	0.93	1.28
15.81	mean	23.3	37.3	124.0	115.0	16.7	14.0	8.7	9.0	36.7	44.3
	MF	1.04	1.00	1.15	1.02	1.19	1.14	0.87	0.90	0.8	1.36
5	mean	24.0	32.3	123.3	114.7	15.3	14.0	13.3	8.0	34.7	43.0
	MF	1.07	0.87	1.15	1.02	1.10	1.14	1.33	0.80	0.76	1.32
NPD 4 µg	mean	412.0	-	-	-	-	-	-	-	-	-
	MF	18.18	-	-	-	-	-	-	-	-	-
2AA 2 µg	mean	-	2538.7	-	2400.0	-	214.0	-	201.3	-	-
	MF	-	81.89	-	21.82	-	18.88	-	25.17	-	-
2AA 50 µg	mean	-	-	-	-	-	-	-	-	-	197.3
	MF	-	-	-	-	-	-	-	-	-	6.10
SAZ 2 µg	mean	-	-	1210.7	-	868.0	-	-	-	-	-
	MF	-	-	11.24	-	62.00	-	-	-	-	-
9AA 50 µg	mean	-	-	-	-	-	-	387.3	-	-	-
	MF	-	-	-	-	-	-	34.18	-	-	-
MMS 2 µL	mean	-	-	-	-	-	-	-	-	949.3	-
	MF	-	-	-	-	-	-	-	-	20.79	-

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.