Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 09 2016 - October 28 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

EC Number:

807-888-6

Cas Number:

110621-40-4

Molecular formula:

Al10.33Mg0.67Na1.67O17

IUPAC Name:

Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 10978-52 H_K


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dry storate
- Stability under test conditions: given
- Solubility and stability of the test substance in the solvent/vehicle: given

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final dilution of a dissolved solid, stock liquid or gel: suspention in ultrapure water

Method

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

First experiment (standard plate test): 0; 33; 100; 333; 1000; 3000 and 6000 μg/plate (top dose was selected based on purity of the test substance)
Second experiment (preincubation test): 0; 10; 33; 100; 333; 1000 and 3000 μg/plate (top dose limited by bacterioxicity)

Vehicle / solvent:

ultrapure water

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: With S9 mix: 2-aminoanthracene (2-AA); without S9 mix: N-methyl-N'-nitro-N-nitrosoguianidine (MNNG)

Details on test system and experimental conditions:

Standard plate test
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Controls

Negative controls / Vehicle controls
Each experiment included negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).
• Sterility control
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.
• Vehicle control
The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

Positive controls
The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
with S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA
The stability of the selected positive controls was well-defined under the selected culture conditions, since they were well-established reference mutagens.

Evaluation criteria:

Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: Salmonella TA 1535, TA 100, TA 1537, TA 98, E. coli

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3000-6000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Standard plate test without metabolic activation:

Strain	Test group	Dose ((µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	12.3	2.3	-	11, 11, 15
	Test item	33	13.3	1.2	1.1	14, 14, 12
		100	12.0	1.7	1.0	13, 10, 13
		333	15.0	4.6	1.2	20, 11, 14
		1000	13.3	2.1	1.1	15, 14, 11
		3000	7.0	1.0	0.6	7 B, 6 B, 8 B
		6000	6.7	1.5	0.5	7 B, 8 B, 5 B
	MNNG	5.0	5152.3	224.5	417.8	5319, 4897, 5241
TA 100	Water	-	103.0	12.0	-	91, 103, 115
	Test item	33	126.7	6.7	1.2	125, 134, 121
		100	117.0	13.9	1.1	124, 101, 126
		333	98.7	11.4	1.0	108, 102, 86
		1000	124.7	4.7	1.2	121, 130, 123
		3000	86.3	8.7	0.8	79 B, 84 B, 96 B
		6000	74.7	8.1	0.7	71 B, 69 B, 84 B
	MNNG	5.0	4218.3	128.0	41.0	4347, 4091, 4217
TA 1537	Water	-	11.3	2.1	-	13, 9, 12
	Test item	33	9.3	3.2	0.8	8, 7, 13
		100	9.0	1.0	0.8	10, 8, 9
		333	7.7	2.1	0.7	7, 6, 10
		1000	8.7	2.1	0.8	8, 11, 7
		3000	6.7	4.0	0.6	11 B, 6 B, 3 B
		6000	7.3	1.5	0.6	6 B, 7 B, 9 B
	AAC	100	1350.3	267.8	119.1	1274, 1129, 1648
TA 98	Water	-	25.0	7.0	-	22, 20, 33
	Test item	33	29.0	5.6	1.2	34, 23, 30
		100	22.0	4.6	0.9	17, 26, 23
		333	20.3	4.9	0.8	17, 26, 18
		1000	21.7	5.5	0.9	27, 22, 16
		3000	16.7	4.2	0.7	18 B, 12 B, 20 B
		6000	15.7	5.0	0.6	15 B, 21 B, 11 B
	NOPD	10	1039.0	51.4	41.6	999, 1097, 1021
E. coli	Water	-	29.0	10.8	-	38, 32, 17
	Test item	33	33.0	7.0	1.1	25, 38, 36
		100	33.7	3.8	1.2	31, 38, 32
		333	27.0	1.0	0.9	26, 28, 27
		1000	22.3	3.1	0.8	23, 19, 25
		3000	12.7	2.5	0.4	15 B, 10 B, 13 B
		6000	5.7	2.5	0.2	6 B, 8 B, 3 B
	4-NQO	5	1011.7	10.3	34.9	1009, 1003, 1023

Standard plate test with metabolic activation:

Strain	Test group	Dose(µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	10.7	6.4	-	8, 18, 6
	Test item	33	12.0	1.0	1.1	12, 13, 11
		100	10.7	2.9	1.0	14, 9, 9
		333	12.0	2.6	1.1	15, 11, 10
		1000	11.3	3.2	1.1	10, 15, 9
		3000	8.7	3.5	0.8	5 B, 9 B, 12 B
		6000	7.0	1.0	0.7	8 B, 7 B, 6 B
	2-AA	2.5	519.7	79.8	48.7	595, 528, 436
TA 100	Water	-	110.3	7.6	-	117, 102, 112
	Test item	33	110.7	10.7	1.0	105, 123, 104
		100	110.3	5.1	1.0	116, 109, 106
		333	89.0	1.0	0.8	89, 90, 88
		1000	105.3	10.3	1.0	114, 108, 94
		3000	102.3	12.7	0.9	88 B, 112 B, 107 B
		6000	105.7	7.4	1.0	103 B, 100 B, 114 B
	2-AA	2.5	1233.3	24.9	11.2	1217, 1262, 1221
TA 1537	Water	-	5.7	1.5	-	6, 4, 7
	Test item	33	8.0	1.7	1.4	7, 7, 10
		100	11.0	4.4	1.9	14, 6, 13
		333	10.0	3.6	1.8	7, 14, 9
		1000	11.7	2.1	2.1	14, 11, 10
		3000	4.7	0.6	0.8	5 B, 5 B, 4 B
		6000	4.0	2.0	0.7	6 B, 2 B, 4 B
	2-AA	2.5	154.7	41.7	27.3	111, 194, 159
TA 98	Water	-	24.7	6.5	-	31, 25, 18
	Test item	33	25.3	0.6	1.0	25, 25, 26
		100	24.3	8.7	1.0	17, 34, 22
		333	25.7	4.0	1.0	21, 28, 28
		1000	26.7	6.5	1.1	20, 27, 33
		3000	22.0	8.5	0.9	31 B, 21 B, 14 B
		6000	13.3	2.1	0.5	15 B, 14 B, 11 B
	2-AA	2.5	2188.0	100.5	88.7	2072, 2250, 2242
E. coli	Water	-	23.7	6.7	-	28, 27, 16
	Test item	33	16.7	2.5	0.7	19, 14, 17
		100	17.7	4.0	0.7	22, 14, 17
		333	25.7	2.3	1.1	27, 23, 27
		1000	24.7	10.5	1.0	35, 25, 14
		3000	22.3	5.9	0.9	18 B, 29 B, 20 B
		6000	24.0	1.7	1.0	26 B, 23 B, 23 B
	2-AA	60	318.7	95.9	13.5	329, 409, 218

Preincubation test without metabolic activation:

Strain	Test group	Dose(µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	8.3	3.2	-	12, 7, 6
	Test item	10	6.3	0.6	0.8	6, 6, 7
		33	7.0	3.6	0.8	8, 10, 3
		100	8.3	3.8	1.0	4, 11, 10
		333	9.7	2.1	1.2	12, 8, 9
		1000	7.3	1.5	0.9	7, 9, 6
		3000	6.7	2.3	0.8	8 B, 8 B, 4 B
	MNNG	5.0	2337.0	121.0	280.4	2457, 2339, 2215
TA 100	Water	-	93.0	11.5	-	84, 89, 106
	Test item	10	100.0	14.7	1.1	117, 92, 91
		33	108.3	6.5	1.2	108, 115, 102
		100	100.7	6.4	1.1	98, 108, 96
		333	78.7	9.0	0.8	70, 78, 88
		1000	87.0	2.0	0.9	87, 85, 89
		3000	79.0	4.0	0.8	83 B, 75 B, 79 B
	MNNG	5.0	2234.0	98.0	24.0	2145, 2339, 2218
TA 1537	Water	-	6.3	1.5	-	6, 8, 5
	Test item	10	5.3	1.5	0.8	4, 5, 7
		33	6.3	2.1	1.0	8, 7, 4
		100	7.0	1.0	1.1	7, 6, 8
		333	5.0	1.0	0.8	4, 6, 5
		1000	5.3	1.2	0.8	4, 6, 6
		3000	0.0	0.0	0.0	0 B, 0 B, 0 B
	AAC	100	711.0	133.1	112.3	655, 615, 863
TA 98	Water	-	19.3	2.5	-	17, 19, 22
	Test item	10	22.0	5.2	1.1	25, 16, 25
		33	18.7	1.2	1.0	20, 18, 18
		100	18.3	2.3	0.9	17, 17, 21
		333	20.0	7.2	1.0	22, 12, 26
		1000	21.3	5.1	1.1	27, 20, 17
		3000	23.0	10.4	1.2	28 B, 30 B, 11 B
	NOPD	10	834.3	47.4	43.2	817, 798, 888
E. coli	Water	-	21.7	8.4	-	12, 27, 26
	Test item	10	23.0	4.6	1.1	28, 22, 19
		33	16.3	3.8	0.8	12, 18, 19
		100	17.0	4.6	0.8	21, 12, 18
		333	22.7	5.9	1.0	25, 27, 16
		1000	20.0	7.9	0.9	17, 14, 29
		3000	19.7	6.0	0.9	26 B, 19 B, 14 B
	4-NQO	5	343.7	60.7	15.9	407, 338, 286

Preincubation test with metabolic activation:

Strain	Test group	Dose(µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	11.0	4.6	-	16, 7, 10
	Test item	10	11.0	5.3	1.0	5, 13, 15
		33	9.7	2.1	0.9	8, 9, 12
		100	7.7	2.1	0.7	10, 6, 7
		333	12.0	2.0	1.1	12, 10, 14
		1000	11.7	3.5	1.1	12, 15, 8
		3000	10.3	0.6	0.9	10 B, 11 B, 10 B
	2-AA	2.5	155.7	27.5	14.2	124, 174, 169
TA 100	Water	-	70.0	8.7	-	65, 80, 65
	Test item	10	81.0	8.2	1.2	74, 79, 90
		33	85.0	7.9	1.2	91, 76, 88
		100	80.7	5.1	1.2	85, 82, 75
		333	86.7	3.8	1.2	91, 85, 84
		1000	82.0	6.0	1.2	76, 88, 82
		3000	81.0	3.0	1.2	81 B, 78 B, 84 B
	2-AA	2.5	2209.0	129.7	31.6	2061, 2263, 2303
TA 1537	Water	-	6.3	2.3	-	5, 5, 9
	Test item	10	7.7	3.1	1.2	11, 7, 5
		33	8.7	4.0	1.4	8, 13, 5
		100	5.3	2.3	0.8	4, 8, 4
		333	7.3	1.2	1.2	6, 8, 8
		1000	7.7	3.1	1.2	5, 7, 11
		3000	5.7	1.2	0.9	5 B, 5 B, 7 B
	2-AA	2.5	253.7	31.0	40.1	281, 220, 260
TA 98	Water	-	29.7	4.2	-	31, 25, 33
	Test item	10	28.0	2.0	0.9	28, 26, 30
		33	28.3	5.5	1.0	32, 31, 22
		100	24.0	4.4	0.8	19, 26, 27
		333	27.0	4.6	0.9	26, 23, 32
		1000	31.7	9.5	1.1	21, 39, 35
		3000	36.7	3.2	1.2	33 B, 39 B, 38 B
	2-AA	2.5	3096.3	66.0	104.4	3046, 3171, 3072
E. coli	Water	-	17.3	1.5	-	16, 17, 19
	Test item	10	23.3	9.6	1.3	25, 13, 32
		33	28.3	1.5	1.6	28, 27, 30
		100	20.3	1.5	1.2	20, 19, 22
		333	20.0	2.6	1.2	18, 23, 19
		1000	25.0	7.9	1.4	16, 31, 28
		3000	23.7	2.5	1.4	26 B, 24 B, 21 B
	2-AA	60	384.0	53.0	22.2	445, 358, 349

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that ß’’-Alumina (BASF) is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.