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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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.