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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:
2015-09-21 to 2015-10-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 471 and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(July 1997)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(May 2008)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Beta'' Alumina Powder
- Physical state: Fine white powder
- Analytical purity: 100% (from supplied MSDS)
- Lot/batch No.: 767

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient medium with 8 g Nutrient Broth and 5 g NaCl per litre. A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre-Experiment for Toxicity (TA 98 and TA 100): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment I (all tester strains): 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
Experiment II (all tester strains): 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: phosphate buffer
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Plate-incorporation (Experiment I): 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), 2000 µL Overlay agar were mixed in a test tube and poured over the surface of a minimal agar plate.
- Pre-incubation (Experiment II): 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: three (in one case, Experiment I TA 98 31.6µg, only two plates were evaluated)

Evaluation criteria:
Criteria of Validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data range (2012 -2014)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed in all tester strains used in experiment I and II at the highest concentrations of 316 and 1000 µg/plate (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment, there was strong precipitation at concentrations of 316 µg/plate and higher. No signs of toxicity were observed at concentrations up to the limit dose of 5000 µg/plate. The concentrations evaluated in the main experiments were chosen on the basis of these results.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Any other information on results incl. tables

Table 1: Results Pre-Experiment

Substance

Dose

(µg/plate)

TA 98

Mutation Factor [toxicity]*

TA 98

Mutation Factor [toxicity]*

 

 

without S9

with S9

without S9

with S9

Solvent Control

(Phosphate Buffer)

 

1.0

1.0

1.0

1.0

4-NOPD

10.0

6.8

-

-

-

NaN3

10.0

-

-

7.0

-

2-AA

2.50

-

40.3

-

23.3

Test Item

3.16

1.0

0.9

0.9

1.3

10.0

1.2

0.9

1.0

1.3

31.6

1.1

0.9

0.9

1.1

100

1.0

1.0

0.9

1.5

316 P

1.1

0.9

0.8

1.2

1000 P

1.1

1.1

1.1

1.4

2500 P

1.0

0.9

1.0

1.3

5000 P

0.9

1.1

0.9

1.1

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

P: Precipitation

 

Table 2: Results Experiment I(Plate-incorporation Test) TA 98

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Mean

SD

Mean

SD

-S9

+S9

A. dest.

 

41

5.9

55

3.5

0.9

0.9

Phosphate Buffer

 

45

2.6

59

4.6

1.0

1.0

Test Item

3.16 µg

45

4.4

54

9.1

1.0

0.9

10.0 µg

53

8.2

54

4.0

1.2

0.9

31.6 µg

48

4.2

53

2.6

1.1

0.9

100 µg

43

5.7

58

7.8

1.0

1.0

316 µg

51

5.1

51

8.7

1.1

0.9

1000 µg

49

5.5

65

7.6

1.1

1.1

4-NOPD

10 µg

305

94.9

-

-

6.8

-

2-AA

2.5 µg

-

-

2363

188.8

-

40.3

Table 3: Results Experiment I(Plate-incorporation Test)TA 100

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Mean

SD

Mean

SD

-S9

+S9

A. dest.

 

91

6.7

108

3.5

0.9

1.2

Phosphate Buffer

 

106

9.6

92

20.2

1.0

1.0

Test Item

3.16 µg

99

4.5

120

5.3

0.9

1.3

10.0 µg

101

23.2

116

21.9

1.0

1.3

31.6 µg

100

5.1

104

17.4

0.9

1.1

100 µg

93

19.1

135

34.0

0.9

1.5

316 µg

90

8.0

108

15.0

0.8

1.2

1000 µg

116

6.1

125

9.6

1.1

1.4

NaN3

10 µg

744

78.6

-

-

7.0

-

2-AA

2.5 µg

-

-

2140

435.2

-

23.3

Table 4: Results Experiment I(Plate-incorporation Test)TA 1535

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Mean

SD

Mean

SD

-S9

+S9

A. dest.

 

14

2.0

8

1.5

1.0

1.1

Phosphate Buffer

 

14

8.1

7

4.0

1.0

1.0

Test Item

3.16 µg

19

3.2

10

2.5

1.4

1.3

10.0 µg

18

2.9

7

2.3

1.3

0.9

31.6 µg

13

3.2

11

3.8

0.9

1.5

100 µg

11

3.8

8

4.6

0.8

1.1

316 µg

16

1.7

9

2.3

1.2

1.3

1000 µg

19

8.0

10

0.0

1.4

1.4

NaN3

10 µg

979

159.5

-

-

71.6

-

2-AA

2.5 µg

-

-

192

22.3

-

26.2

Table 5: Results Experiment I(Plate-incorporation Test)TA 1537

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Mean

SD

Mean

SD

-S9

+S9

A. dest.

 

6

1.0

6

2.1

2.0

1.3

Phosphate Buffer

 

3

1.7

4

1.2

1.0

1.0

Test Item

3.16 µg

3

1.0

5

1.2

1.0

1.2

10.0 µg

3

1.0

5

1.2

1.0

1.1

31.6 µg

6

2.1

4

1.5

1.9

1.0

100 µg

8

5.7

5

1.0

2.6

1.2

316 µg

5

2.1

6

1.5

1.6

1.3

1000 µg

7

1.5

5

0.6

2.2

1.2

4-NOPD

10 µg

61

4.0

-

-

20.3

-

2-AA

2.5 µg

-

-

230

23.9

-

53.0

Table 6: Results Experiment I(Plate-incorporation Test)TA 102

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Mean

SD

Mean

SD

-S9

+S9

A. dest.

 

298

27.8

415

17.4

1.1

1.0

Phosphate Buffer

 

273

22.6

416

44.5

1.0

1.0

Test Item

3.16 µg

285

35.9

422

15.7

1.0

1.0

10.0 µg

298

34.0

346

58.9

1.1

0.8

31.6 µg

300

4.5

382

74.3

1.1

0.9

100 µg

246

16.5

348

44.3

0.9

0.8

316 µg

298

28.6

388

11.0

1.1

0.9

1000 µg

325

13.1

462

26.5

1.2

1.1

MMS

10 µg

2036

286.8

-

-

7.5

-

2-AA

2.5 µg

-

-

1086

47.4

-

2.6

SD: Standard-deviation

B: Background lawn reduced

N: No background lawn

P: Percipitation

C: Contamination

Mutation factor = mean revertants (test item) / mean revertants (vehicle control)

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Sodium Beta“ Alumina, Lithium Doped (HT767) did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Sodium Beta“ Alumina, Lithium Doped (HT767) is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of Sodium Beta“ Alumina, Lithium Doped (HT767) for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

Experiment II:

1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

Precipitation was observed in all tester strains used in experiment I and II (with and without

metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Sodium Beta“ Alumina, Lithium Doped (HT767) at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.