Beryllium

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:

2008-11-28 to 2009-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-compliant study under GLP without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

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:

Beryllium

EC Number:

231-150-7

EC Name:

Beryllium

Cas Number:

7440-41-7

Molecular formula:

Be

IUPAC Name:

beryllium

Details on test material:

- Name of test material (as cited in study report): Beryllium Metal Powder
- Substance type: Metal
- Physical state: solid, powdered
- Analytical purity: 99.4 %
- Lot/batch No.: O-30 H grade/Blend # 051106
- Expiration date of the lot/batch: 2050-12-31
- Storage condition of test material: dry

The test item was extracted in 0.9 % saline for 72 h in the dark at 37 °C under non-abrasive conditions. Particulate matter was removed by centrifugation before incubation of the bacteria.

Method

Target gene:

TA 1537 his C 3076; rfa-; uvrB-
TA 98 his D 3052; rfa-; uvrB-;R-factor
TA 1535 his G 46; rfa-; uvrB-
TA 100 his G 46; rfa-; uvrB-;R-factor
WP2 uvrA trp-; uvrA-

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
Precultures: 8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt), 5 g NaCl (MERCK, D-64293 Darmstadt)
Agar: 6.0 g MERCK Agar Agar
6.0 g NaCl
10.5 mg L-Histidine*HCl*H2O
12.2 mg Biotin


- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

2.5, 10, 20, 40, 60, 80, 100 % test item extract (see "Details on Test Material")

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiol. saline

- Justification for choice of solvent/vehicle: due to insolubility of beryllium metal in aqueous media at relevant pH for genotoxicity testing, extracts had to be prepared simulating lung conditions as the most relevant route of exposure

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

physiol. saline

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: and sodium azide; 2-aminoanthracene; 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (Exp. I), in agar (plate incorporation; Exp. II);


DURATION
- Preincubation period: 60 min.
- Selection time (if incubation with a selection agent): 48 h


SELECTION AGENT (mutation assays): ready-made selection agar plates from Merck, suitable for the individual strains


NUMBER OF REPLICATIONS: 3 replicates for each strain and dose level, in each experiment



DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertants

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.

Statistics:

none applied

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative 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

Table 1:Number of revertants per plate (mean of 3 plates); Experiment I

	TA 1535			TA 1537			TA 98
Conc. [% extract]	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	15	19	n	12	11	n	125	140	n
2.5	15	20	n	10	10	n	135	144	n
5	16	16	n	14	12	n	126	139	n
10	18	19	n	12	12	n	134	148	n
20	16	17	n	11	11	n	115	132	n
40	13	17	n	10	13	n	122	145	n
60	15	18	n	6	12	n	132	141	n
80	15	15	n	10	11	n	130	142	n
100	16	14	n	10	12	n	125	125	n
Positive control	1998	373	n	83	258	n	2125	2418	n

	TA 100			WP2 uvrA
Conc. [% extract]	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	125	140	n	58	60	n
2.5	135	144	n	65	66	n
5	126	139	n	63	67	n
10	134	148	n	62	68	n
20	115	132	n	59	65	n
40	122	145	n	57	58	n
60	132	141	n	67	61	n
80	130	142	n	57	62	n
100	125	125	n	61	62	n
Positive control	2125	2418	n	1492	181	n

*solvent control with 0.9% NaCl

 

Table 1:Number of revertants per plate (mean of 3 plates); Experiment II

	TA 1535			TA 1537			TA 98
Conc. [% extract]	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	15	21	n	17	19	n	22	31	n
10	11	19	n	14	19	n	24	34	n
20	13	22	n	15	14	n	22	38	n
40	14	15	n	16	17	n	25	36	n
60	14	19	n	15	17	n	21	31	n
80	13	20	n	12	18	n	19	31	n
100	17	19	n	19	18	n	20	34	n
Positive control	1840	237	n	105	184	n	377	1324	n

	TA 100			WP2 uvrA
Conc. [% extract]	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	156	178	n	52	53	n
10	159	196	n	55	64	n
20	157	162	n	58	56	n
40	150	172	n	53	61	n
60	161	168	n	47	55	n
80	163	171	n	53	63	n
100	142	170	n	53	64	n
Positive control	1906	1550	n	284	248	n

*solvent control with 0.9% NaCl

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No genotoxic potentail of beryllium metal powder was identified in a bacterial reverse mutation assay in absence and presence of metabolic activation.

Executive summary:

The mutagenic potential of beryllium metal powder was investigated in an Ames test according to OECD guideline 471. Briefly, bacteria (S. typhimurium TA1535, TA1537, TA98, TA 100 and E. coli WP2 uvrA) were exposed to the test item in presence and absence of metabolic activation (rat liver S9) and grown on selective agar plates, eliminating bacteria without mutations. The colonies formed from surviving (= mutant) bacteria were counted.

Beryllium metal was - due to its insolubility in aqueous media at neutral pH - extracted in physiological saline and the extracts were applied to expose the bacteria. In the first expereiment the pre-incubation and in the second experiment the plate incorporation method were used.

No increases of mutant numbers were observed in treated groups compared to control. The test item was non-mutagenic in the bacterial reverse mutation assay under the test conditions.