Samarium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2 May 2012 to 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The surface of samarium metal oxidises on contact with air to form an outer layer of samarium oxide. It is therefore considered appropriate to read across information from samarium oxide to the metal where testing on the metal is not technically possible.

Cross-reference

Reason / purpose for cross-reference:

other: read-across target

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

The surface of samarium metal oxidises on contact with air to form an outer layer of samarium oxide. It is therefore considered appropriate to read across information from samarium oxide to the metal where testing on the metal is not technically possible.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and 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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Samarium (III) oxide

EC Number:

235-043-6

EC Name:

Samarium (III) oxide

Cas Number:

12060-58-1

Molecular formula:

O3Sm2

IUPAC Name:

Samarium (III) oxide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellowish powder
- Storage conditions: room temperature (20 ± 5°C)

Specific details on test material used for the study:

As the test material is not sufficiently soluble in any acceptable vehicle for use in an Ames test, the supernatant of suspensions were used. The test material was weighed directly for all tested concentrations and suspended in sterile aqua demin. All suspensions were stirred during the test.
As no complete dissolution was possible, undissolved particles were visible on the plates.

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).

Species / strain

Species / strain / cell type:

other: S typhimurium TA 97a, TA 98, TA 100, TA 102, and TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First experiment: 0, 51, 157, 498, 1497 and 5007 µg/plate (without and with activation)
Second experiment: 317, 626, 1255, 2508 and 5011 µg/plate (without and with activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine (20 µg/plate) and 2-Amino-anthracene (1 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

EXPERIMENT 1- PLATE INCORPORATION METHOD
100 µL test material suspension, vehicle or positive control was added to 100 µL bacteria suspension and 500 µL S9 mix or 500 µL phosphate buffer (for tests with and without metabolic activation, respectively) and 2 000 µL overlay agar. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

EXPERIMENT 2- PRE-INCUBATION METHOD
100 µL test material suspension, vehicle or positive control was added to 100 µL bacteria suspension and 500 µL S9 mix or 500 µL phosphate buffer (for tests with and without metabolic activation, respectively) and gently vortexed in a test tube and incubated at 37°C for 20 minutes. After pre-incubation 2 000 µL overlay agar was added, the tube gently vortexed and the mixture poured onto the selective agar plate. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

The colonies were counted visually, the numbers were recorded.

Evaluation criteria:

A test material is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and 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

Additional information on results:

EXPERIMENT 1
- Confirmation of the Criteria and Validity: The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.
- Toxicity: No signs of toxicity towards tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- Mutagenicity: No significant increase in the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test material was found not to be mutagenic under the sonditions of this experiment. To verify the result, a second experiment was conducted using the pre-incubation method.

EXPERIMENT 2
- Confirmation of the Criteria and Validity: The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.
- Toxicity: No signs of toxicity towards tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.
- Mutagenicity: No significant increase in the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

The results of the second experiment confirmed the findings of the first experiment.

Any other information on results incl. tables

First Experiment:

The mean revertant values of the four replicates are presented in the following table. Concentrations of the test mateiral are stated as nominal concentrations, as suspensions had to be tested due to the poor solubility of the test material. As no complete dissolution was possible, undissolved particles were visible on the plates.

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	108	116	15	16	87	89	140	146	13	15
	sd	13.1	5.1	3.9	1.4	10.2	3.6	7.9	7.7	1.5	2.6
DMSO	Mean	104	108	12	17	88	91	150	149	15	17
	sd	6.5	13.1	1.7	1.2	6.8	11.4	14.7	10.2	1.7	1.0
Positive Controls	Mean	531	583	245	217	516	559	582	632	212	202
	sd	74	88	15	15	38	28	25	39	21	21
	f(I)	5.11	5.40	20.42	12.76	5.93	6.14	3.88	4.24	16.31	11.88
5007 µg/pl.	Mean	113	118	17	15	79	77	152	144	15	15
	sd	6	4	2	1	6	10	6	9	4	1
	f(I)	1.05	1.02	1.13	0.94	0.91	0.87	1.09	0.99	1.15	1.00
1497 µg/pl.	Mean	117	119	16	15	84	93	145	142	17	15
	sd	3	5	2	1	11	13	10	9	4	2
	f(I)	1.08	1.03	1.07	0.94	0.97	1.04	1.04	0.97	1.31	1.00
498 µg/pl.	Mean	120	121	16	16	77	96	148	137	16	16
	sd	2	2	2	1	8	9	5	6	3	2
	f(I)	1.11	1.04	1.07	1.00	0.89	1.08	1.06	0.94	1.23	1.07
157 µg/pl.	Mean	114	116	16	15	76	93	129	147	15	14
	sd	3	4	2	2	22	16	10	13	4	1
	f(I)	1.06	1.00	1.07	0.94	0.87	1.04	0.92	1.01	1.15	0.93
51 µg/pl.	Mean	120	113	15	16	82	84	144	133	14	14
	sd	3	5	4	1	5	6	8	12	3	1
	f(I)	1.11	0.97	1.00	1.00	0.94	0.94	1.03	0.91	1.08	0.93

Second Experiment:

The mean revertant values of the four replicates are presented in the following table. Concentrations of the test material are stated as nominal concentrations, as suspensions had to be tested due to the poor solubility of the test material. As no complete dissolution was possible, undissolved particles were visible on the plates.

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	116	106	21	20	66	83	135	137	14	14
	sd	15.9	7.5	2.5	2.3	4.3	12.0	18.3	18.2	1.3	3.0
DMSO	Mean	112	109	15	18	70	84	138	146	14	13
	sd	3.0	6.7	2.6	2.9	9.7	5.7	9.2	4.5	2.2	3.4
Positive Controls	Mean	449	301	885	824	683	617	593	692	343	232
	sd	75	38	47	53	82	98	36	67	80	8
	f(I)	4.01	2.76	59.00	45.78	10.35	7.35	4.30	4.74	24.50	17.85
5011 µg/pl.	Mean	101	103	16	17	114	102	138	134	15	16
	sd	7	7	2	4	6	7	3	15	2	2
	f(I)	0.87	0.97	0.76	0.85	1.73	1.23	1.02	0.98	1.07	1.14
2508 µg/pl.	Mean	102	101	15	19	92	100	132	141	15	16
	sd	5	12	2	2	19	4	5	2	1	3
	f(I)	0.88	0.95	0.71	0.95	1.39	1.20	0.98	1.03	1.07	1.14
1255 µg/pl.	Mean	112	96	13	18	85	100	151	146	14	14
	sd	8	4	3	2	10	7	7	9	1	3
	f(I)	0.97	0.91	0.62	0.90	1.29	1.20	1.12	1.07	1.00	1.00
626 µg/pl.	Mean	93	98	15	14	87	96	151	129	16	14
	sd	3	6	1	2	6	8	8	4	2	3
	f(I)	0.80	0.92	0.71	0.70	1.32	1.16	1.12	0.94	1.14	1.00
317 µg/pl.	Mean	97	93	15	14	84	105	136	145	16	14
	sd	4	3	2	3	11	9	6	10	2	1
	f(I)	0.84	0.88	0.71	0.70	1.27	1.27	1.01	1.06	1.14	1.00

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The mutagenic potential of the test material was investiagted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions using the Bacterial Reverse Mutation Assay.

Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels both with and without the addition of a rat liver homogenate metabolising system.

Under the conditions of the study, the test material was considered not mutagenic. Also, the test material did not show any cytotoxicity towards the bacteria.

As no complete dissolution was possible, undissolved particles were visible on the plates. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were definitely increased in comparison with the negative controls, as well as showing mutagenic potential of the diagnostic mutagens.

The findings of the study are therefore considered to be valid.