undecaaluminium sodium heptadecaoxide, lithium doped

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(July 1997)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(May 2008)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

undecaaluminium sodium heptadecaoxide, lithium doped

EC Number:

629-817-6

Cas Number:

1236215-65-8

Molecular formula:

Na (1+2x) Li x Al (11-x) O17 with x between 0.3 and 0.35

IUPAC Name:

undecaaluminium sodium heptadecaoxide, lithium doped

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.