Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
In vitro mutagenicity assessment of aluminium oxide nanomaterials using the Salmonella/microsome assay
Author:
Balasubramanyam, A., et al.
Year:
2010
Bibliographic source:
Toxicology in Vitro 24: 1871-1876

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
2020
Deviations:
yes
Remarks:
No test concentration (< 5 µg/plate) was insoluble in the final treatment mixture. No justificatin provided for not performing a confirmation of negative results; no data, if the efficacy of the S9 mix was chararcterized by a second mutagen.
GLP compliance:
not specified
Remarks:
The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium oxide
EC Number:
215-691-6
EC Name:
Aluminium oxide
Cas Number:
1344-28-1
Molecular formula:
Al2O3
IUPAC Name:
aluminium oxide

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: 97a
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- method of preparation of S9 mix: The S9 mix was prepared just before use by adding 900 µL of co-factors (5.2 mM D-glucose-6-phosphate, 4.4 mM nicotinamide adenine dinucleotide phosphate, 18 mM MgCl2, 28 mM KCl, 90 mM Na2HPO4.H2O, 8 mM NaH2PO4.H2O) and 100 µL S9 fraction.
Test concentrations with justification for top dose:
0.02, 0.04, 0.075, 0.15, 0.3, 0.6, 1.25 and 2.5 µg/plate with and without metabolic activation in all strains

The test material precipitated at concentrations higher than 2.5 µg/plate. Thus, this concentration was chosen as maximum one to test.
Vehicle / solvent:
- Vehicle used: DMSO/H2O 1:1

The test material was suspended in the vehicle and ultrasonicated for 10 min.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO/H2O 1:1 (v/v)
True negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: sextuplicates
- Number of independent experiments : single experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approx. 1E+09 bacterial cells/mL
- Test substance added in: preincubation mixture

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min at 37 °C
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies or a clearing of the background lawn in comparison with control plates

Evaluation criteria:
A positive response in the test was defined as an increase (at least twofold above the control) in His+/wild revertant colonies in every strain.
Statistics:
Mean values and standard deviation were calculated. The one-way analysis of variance (one-way ANOVA), followed by Dunnett’s multiple comparison post test, was used to verify the significance of a positive response. A P value < 0.05 was considered statistically significant.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
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:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
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:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
STUDY RESULTS
- Concurrent positive control data : The positive control substances (SA, 2NF, 9AA, mitomycin C and 2AA) at µg levels increased the number of revertant colonies by several fold compared to the control, revealing the sensitivity of the system to detect a mutagenic effect.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : Even at the highest tested concentration no increase in the number of revertants was detected and thus no concentration-response relationship is evident.
- Statistical analysis; P-value : All positive control substances resulted in P < 0.01 vs. control (ANOVA).


Ames test:
- Signs of toxicity : All concentrations tested did not show any statistically significant decrease in the number of revertant colonies compared to the control and thus, they resulted in lack of cytotoxicity.
- Individual plate counts : no data
- Mean number of revertant colonies per plate and standard deviation : please refer to table 1


HISTORICAL CONTROL DATA: no data

Any other information on results incl. tables

Table 1. Test results for Al2O3-bulk

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate (n=6)

± standard deviation

TA100

TA1535

TA98

TA97a

TA102

+

DMSO (50 µL/plate)

153.3 ± 14.9

12.6 ± 5.6

23.3 ± 4.5

178.3 ± 11.5

320.6 ± 21.5

+

DMSO/H2O 1:1 (v/v)

146.4 ± 16.2

13.3 ± 4.9

26.1 ± 5.3

169.2 ± 10.2

311.2 ± 22.2

+

20

154.6 ± 10.9

12.4 ± 3.2

24.3 ± 4.5

177.1 ± 14.5

319.0 ± 23.1

+

40

151.3 ± 9.0

13.4 ± 3.8

26.5 ± 4.0

178.6 ± 14.2

320.8 ± 21.3

+

75

154.7 ± 10.5

11.9 ± 5.8

24.6 ± 4.1

179.6 ± 11.8

322.1 ± 19.3

+

150

157.3 ± 10.1

12.6 ± 3.7

26.7 ± 4.8

176.2 ± 15.1

323.6 ± 21.1

+

300

152.2 ± 9.7

12.3 ± 6.1

24.1 ± 3.6

175.3 ± 11.5

320.3 ± 39.1

+

600

161.3 ± 10.5

13.3 ± 4.9

26.9 ± 6.2

175.3 ± 16.4

320.6 ± 27.2

+

1250

166.3 ± 11.2

13.9 ± 5.1

25.3 ± 5.5

171.2 ± 16.1

327.3 ± 24.5

+

2500

169.7 ± 9.8

13.2 ± 4.4

26.6 ± 4.4

174.5 ± 14.8

323.4 ± 22.3

+

Positive controls

2AA

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

695.6 ± 32.9*

432.4 ± 27.9*

208.9 ± 17.2*

830.6 ± 36.4*

1212 ± 82.5*

-

DMSO (50 µL/plate)

102.3 ± 17.5

16.6 ± 5.0

25.3 ± 3.1

174.0 ± 19.8

254.0 ± 25.0

-

DMSO/H2O 1:1 (v/v)

104.6 ± 10.2

14.4 ± 3.9

23.1 ± 3.3

169.6 ± 15.5

241.2 ± 22.2

-

20

106.0 ± 14.3

18.0 ± 3.0

26.1 ± 3.1

171.6 ± 14.

241.1 ± 20.2

-

40

99.3 ± 14.2

17.2 ± 5.2

23.3 ± 2.5

184.0 ± 24.5

264.0 ± 21.6

-

75

112.6 ± 8.5

15.0 ± 5.1

26.3 ± 3.5

171.3 ± 19.6

251.0 ± 25.7

-

150

108.3 ± 10.5

16.0 ± 2.6

23.3 ± 3.1

180.0 ± 22.0

267.3 ± 31.7

-

300

116.0 ± 11.3

16.6 ± 4.1

25.1 ± 3.7

172.3 ± 19.0

268.6 ± 28.8

-

600

126.0 ± 13.7

18.3 ± 3.5

24.3 ± 3.5

177.3 ± 17.7

277.3 ± 29.1

-

1250

114.4 ± 12.3

17.7 ± 2.9

25.2 ± 2.8

180.1 ± 16.8

268.6 ± 30.1

-

2500

121.4 ± 14.2

19.1 ± 3.1

26.7 ± 3.3

182.1 ± 15.4

271.4 ± 33.1

-

Positive controls

SA

SA

2NF

9AA

Mitomycin C

Mean No. of colonies/plate

± SD

483.6 ± 53.5*

323.3 ± 26.5*

95.3 ± 8.6*

625.6 ± 43.4*

1101 ± 84.02*

SA = sodium azide

9AA = 9-aminoacridine

2NF = 2-nitrofluorene

2AA = 2-aminoanthracene

* P < 0.01 vs. Control (ANOVA)

Applicant's summary and conclusion

Conclusions:
Al2O3-bulk was negative in the Ames test in the S. typhimurium strains TA 97a, TA 98, TA 100, TA 1535 and TA102 up to and including a concentration of 2500 µg/plate. No cytotoxicity nor precipitation was noted in any of the tested concentrations.