Dierbium trioxide

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:

22 August 2012 - 22 October 2012

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 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: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dierbium trioxide

EC Number:

235-045-7

EC Name:

Dierbium trioxide

Cas Number:

12061-16-4

Molecular formula:

Er2O3

IUPAC Name:

dierbium(3+) trioxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: pink powder
- Storage conditions: room temperature in the dark under nitrogen

Method

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test
Experiment 1: 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
0.1 mL aliquots of the bacterial cultures were dispensed into sets of test tubes, followed by 2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli added to each 100 mL of top agar), 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation). The contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
The positive and untreated controls were dosed using the standard plate incorporation method described above.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1537 (with and without S9 mix) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA100 (without S9-mix only)

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed with TA100 and WP2uvrA in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.15 to 5000 µg/plate. The test material was not toxic at any concentration both in the absence and presence of S9-mix.


DEFINITIVE STUDY
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The mean number of revertant colonies for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiments 1 and 2, respectively.

The test material caused a visible reduction in the growth of the bacterial background lawns of Salmonella typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No toxicity was noted to any of the remaining bacterial strains at any test material dose level in both experiments in either the absence or presence of S9-mix. The test material was therefore tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix employing each exposure method.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

All of the positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment	Mean number of colonies/plate
	Base-pair Substitution Type			Frameshift Type
	TA100	TA1535	WP2uvrA	TA98	TA1537
1	82	18	26	32	9
2	77	17	31	14	12

 

Table 2 Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - -	Solvent 50 150 500 1500 5000	91 82 85 85 80 70*	16 15 14 17 15 14	26 28 26 30 26 25	28 22 28 26 26 27	17 13 12 15 15 10*
+ + + + + +	Solvent 50 150 500 1500 5000	99 94 102 97 99 84	13 12 10 10 11 12	31 33 31 27 35 26	27 27 27 26 29 27	11 12 11 11 12 7*
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	749	541	267	159	113
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	1281	261	264	176	217

*Sparse bacterial background lawn

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

 

Table 3 Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - - - -	Solvent 5 15 50 150 500 1500 5000	79 77 75 74 71 70 70 70*	17 19 11 21 15 14 16 15	27 24 24 29 26 25 27 29	15 13 13 13 14 11 11 16	12 11 7 10 8 10 10 7*
+ + + + + + + +	Solvent 5 15 50 150 500 1500 5000	85 85 74 82 84 85 76 72	12 12 12 12 10 10 10 11	26 26 27 24 31 24 23 29	26 23 15 20 20 18 18 19	11 8 8 12 9 8 8 6*
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	514	590	484	141	714
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	1455	246	200	223	281

*Sparse bacterial background lawn

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation.

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

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five and seven dose levels, respectively, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate using the plate incorporation method and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate using the pre-incubation method.

The test material caused a visible reduction in the growth of the bacterial background lawns of S. typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No toxicity was noted to any of the remaining bacterial strains at any test item dose level in both experiments in either the absence or presence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. 

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.