Beryllium 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:

2010-03-01 to 2010-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

No deviations with impact on study

Principles of method if other than guideline:

Additional guidelines uses:
"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21, 1997
"Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000
ISO-10993-1; 2009 (E): Biological evaluation of medical devices – Part 1: Evaluation and testing.
ISO-10993-3; 2003 (E): Biological evaluation of medical devices – Part 3: Tests for genotoxicity, carcinogenicity, and reproductive toxicity.
ISO-10993-12, 2007 (E): Biological evaluation of medical devices – Part 12: Sample Preparation and Reference Materials

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Beryllium oxide

EC Number:

215-133-1

EC Name:

Beryllium oxide

Cas Number:

1304-56-9

Molecular formula:

BeO

IUPAC Name:

oxoberyllium

Details on test material:

- Name of test material (as cited in study report): BeO
- Substance type: powder
- Physical state: solid
- Analytical purity: 99.9
- Lot/batch No.: UOX Lot No.1846-B
- Expiration date of the lot/batch: 31.12.2010
- Stability under test conditions: Stable at least until December 31, 2010
- Storage condition of test material: Room temperature

The test item is relatively insoluble in aqueous and organic solvents. The test item was extracted for 72 hours at 37 °C in 0.9 % saline in the dark under non-abrasive shaking according to ISO 10993-12 at a weight/volume ratio of 0.1 g/ml. Particulate metal were removed by centrifugation at ≥ 3000 rpm for a minimum of 10 min. (4.243g/42.4 ml in experiment I, 5.8409g/58.4 ml in experiment II), and the supernatant was used for exposure of bacteria. The pH of the extract was determined after particle removal. The pH value of extract was 5.0, determined in the substitution buffer the pH value was 7 in both experiments. No precipitation of the test item occurred up to the highest investigated dose.

Determination of beryllium content of extracts was done by atomic absorption spectrometry and compared against a beryllium atomic absorption standard. Each sample was analyzed directly by using a graphite furnace coupled to an atomic absorption spectrometer. Where necessary, the samples were further diluted with bidistilled water into the calibration range. The results are reported under 'test concentrations'.

Method

Target gene:

His, Trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

10; 20; 40; 60; 80; and 100 % of the extract. Analytical determination of BeO content was done on two separate days:
10 %: 1.75 and 2.893 μg/l and
20 % :2.95 and 5.855 μg/l
40 %: 5.54 and 13.4 μg/l
60 %: 10.77 and 15.4 μg/l
80 %: 15.2 and 27.5 μg/l
100 %: 19.67 and 35.14 μg/l

Vehicle / solvent:

0.9 % Saline

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Sodium Azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate

Remarks:

With and without metabolic activation

Details on test system and experimental conditions:

For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates (Experiment I):
200 µL Test solution at each dose level, solvent (negative control) or
100 µL reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
In the pre-incubation assay (Experiment II) 200 µl test solution, 100 µl reference mutagen solution (positive control), 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark. The amount of S9 supernatant was 10 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment I Plate incorporation test

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	NaCl 0.9 %			14 ± 1	15 ± 2	34 ± 7	143 ± 5	52 ± 5
	Untreated			12 ± 1	15 ± 2	27 ± 4	150 ± 14	47 ± 3
	BeO Powder	10 %		13 ± 4	16 ± 5	33 ± 4	125 ± 8	56 ± 8
		20 %		13 ± 3	14 ± 1	34 ± 10	139 ± 4	49 ± 5
		40 %		15 ± 3	12 ± 2	36 ± 7	138 ± 7	48 ± 4
		60 %		16 ± 2	13 ± 1	30 ± 1	126 ± 11	47 ± 6
		80 %		14 ± 2	15 ± 5	29 ± 2	132 ± 8	47 ± 5
		100 %		15 ± 3	14 ± 1	36 ± 5	132 ± 5	51 ± 3
	NaN3	10 µg		1821 ± 222			2166 ± 130
	4-NOPD	10 µg				403 ± 26
	4-NOPD	50 µg			122 ± 19
	MMS	3.0 µl						644 ± 50
With Activation	NaCl 0.9 %			20 ± 1	23 ± 1	48 ± 4	129 ± 7	63 ± 12
	Untreated			20 ± 3	21 ± 4	50 ± 7	126 ± 7	58 ± 12
	BeO Powder	10 %		18 ± 2	20 ± 3	44 ± 1	125 ± 11	59 ± 7
		20 %		20 ± 3	21 ± 0	47 ± 5	133 ± 5	61 ± 8
		40 %		23 ± 2	23 ± 3	45 ± 6	117 ± 1	60 ± 7
		60 %		19 ± 4	22 ± 1	44 ± 6	114 ± 6	51 ± 8
		80 %		19 ± 3	23 ± 4	46 ± 3	115 ± 7	57 ± 9
		100 %		20 ± 4	22 ± 2	48 ± 1	122 ± 7	60 ± 7
	2-AA	2.5 µg		458 ± 45	427 ± 11	3390 ± 143	2978 ± 58
	2-AA	10.0 µg						376 ± 14

Experiment II Preincubation test

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	NaCl 0.9 %			10 ± 2	14 ± 3	25 ± 7	153 ± 6	46 ± 10
	Untreated			16 ± 3	13 ± 3	29 ± 2	171 ± 20	45 ± 11
	BeO Powder	10 %		11 ± 3	15 ± 4	26 ± 6	157 ± 14	46 ± 5
		20 %		12 ± 1	16 ± 3	28 ± 7	153 ± 12	47 ± 5
		40 %		11 ± 5	13 ± 1	26 ± 4	155 ± 4	45 ± 5
		60 %		10 ± 3	14 ± 3	24 ± 4	158 ± 13	43 ± 2
		80 %		14 ± 1	14 ± 4	23 ± 2	162 ± 4	43 ± 6
		100 %		11 ± 3	16 ± 1	30 ± 5	162 ± 7	48 ± 1
	NaN3	10 µg		1387 ± 72			1693 ± 121
	4-NOPD	10 µg				392 ± 17
	4-NOPD	50 µg			116 ± 21
	MMS	3.0 µl						499 ± 17
With Activation	NaCl 0.9 %			15 ± 3	20 ± 4	48 ± 6	140 ± 12	53 ± 5
	Untreated			18 ± 3	18 ± 6	53 ± 1	105 ± 7	56 ± 2
	BeO Powder	10 %		16 ± 2	20 ± 3	49 ± 5	133 ± 17	52 ± 4
		20 %		15 ± 5	20 ± 5	44 ± 1	145 ± 19	52 ± 5
		40 %		18 ± 3	22 ± 6	43 ± 2	139 ± 2	55 ± 1
		60 %		16 ± 3	22 ± 6	52 ± 2	122 ± 3	54 ± 7
		80 %		16 ± 1	20 ± 3	50 ± 7	141 ± 12	54 ± 5
		100 %		18 ± 2	21 ± 2	45 ± 7	143 ± 9	50 ± 4
	2-AA	2.5 µg		245 ± 23	283 ± 26	2747 ± 251	1691 ± 107
	2-AA	10.0 µg						396 ± 33

Applicant's summary and conclusion

Conclusions:

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

BeO powder extracted into 0.9 % saline was not genotoxic under the conditions of this study.

Executive summary:

BeO powder was extracted into 0.9 % saline for 72 hours at 37 °C in the dark. No precipitation of BeO occurred up to the highest investigated dose. The BeO extract was tested in the S. typhimurium strains TA 98, TA100, TA 1535 and TA 1537 and the E. coli strain WP2 uvrA at the following concentrations: 10; 20; 40; 60; 80; and 100 % of the extract. Both a plate incorporation assay (experiment I) and a preincubation assay (experiment II) was performed. Positive controls requiring/not requiring metabolic activation was used. No increase in the number of revertant colonies were observed in experiment I or experiment II. It is concluded that BeO powder extracted into 0.9 % saline was not genotoxic under the conditions of this study.