Bis(acetato-O)hydroxyaluminium

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:

10 January 2017 - 02 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminum hydroxide diacetate monohydrate

Cas Number:

80164-67-6

Molecular formula:

C4H7AlO5 H2O

IUPAC Name:

Aluminum hydroxide diacetate monohydrate

Test material form:

solid

Method

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:

S9 mix induced by ß-Naphthoflavone/Phenobarbital in livers of rats

Test concentrations with justification for top dose:

1st series: 5.0, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate (with an without S9 mix)
2nd series: 500, 889, 1580, 2810, 5000 µg/plate (with an without S9 mix)
top dose: maximum recommended concentration (according to OECD 471)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Based on the available information from the preliminary solubility test. DMSO showed best performance and was thus used for this experiment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-aminoanthracene, daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 to 3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 30%

Evaluation criteria:

A test material was to be defined as negative or non-mutapcnic in this assay if
- The assay was to be considered valid, and
- "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
- a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
- "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test 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:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material precipitate was observed on the plates at concentrations ≥ 50 ug/plate.

Any other information on results incl. tables

Table 1: Summary 1st series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		39 ± 6	112 ± 12	24 ± 6	8 ± 2	31 ± 6
	Art 101071	5.00	47 ± 13	101 ± 13	25 ± 6	9 ± 6	27 ± 3
		15.8	40 ± 2	110 ± 5	29 ± 1	5 ± 5	32 ± 6
		50.0	43 ± 14^S B	124 ± 16^SB	30 ± 8^S B	5 ± 1^S B	26 ± 5^S B
		158	39 ± 10^S B	101 ± 7^S B	23 ± 2^S B	7 ± 2^S B	29 ± 6^S B
		500	31 ± 6^S E	109 ± 1^S E	21 ± 4^S E	5 ± 3^S E	29 ± 8^S E
		1580	36 ± 4^S E	97 ± 9^S E	22 ± 3^S E	7 ± 2^S E	24 ± 8^S E
		5000	32 ± 2^S E	95 ± 11^S E	12 ± 1^S E	4 ± 1^S E	30 ± 8^S E
	DAUN	1.00	314 ± 45
	NaN3	2.00		1785 ± 9	1078 ± 81
	9-AA	50.0				551 ± 41
	NQO	2.00					1692 ± 65
With Activation	DMSO		45 ± 7	118 ± 13	25 ± 7	10 ± 3	35 ± 5
	Art. 101071	5.00	43 ± 6	127 ± 24	26 ± 6	9 ± 4	40 ± 7
		15.8	46 ± 11^C	114 ± 27	24 ± 1	8 ± 2	37 ± 1
		50.0	42 ± 8^S B	122 ± 7^S B	24 ± 5^S B	9 ± 4^S B	39 ± 4^S B
		158	43 ± 7^S B	132 ± 5^S B	22 ± 2^S B	8 ± 5^S B	32 ± 11^S B
		500	41 ± 11^S E	126 ± 4^S E	22 ± 6^S E	7 ± 2^S E	32 ± 10^S E
		1580	51 ± 10^E S	122 ± 2^E S	26 ± 11^E S	9 ± 6^E S	33 ± 4^E S
		5000	46 ± 3^S E	105 ± 12^SE	30 ± 6^S E	1 ± 1^S E	41 ± 5^S E
	2-AA	2.00	2911 ± 509	3327 ± 228
	2-AA	5.00			142 ± 11	272 ± 36
	2-AA	10.0					415 ± 27

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide,S: Plated as suspension, B: Precipitation at beginning of experiment, E: Precipitation until end of experiment, C: Contaminated

Table 2: Summary 2nd series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		37 ± 7	103 ± 8	26 ± 6	8 ± 4	29 ± 7
	Art 101071	500	39 ± 4^S B	118 ± 7^S B	19 ± 4^S B	7 ± 2^S B	27 ± 7^S B
		889	44 ± 1^S E	110 ± 8^S E	25 ± 5^S E	7 ± 3^S E	31 ± 1^S E
		1580	43 ± 7^S E	102 ± 11^SE	24 ± 3^S E	8 ± 4^S E	29 ± 1^S E
		2810	29 ± 3^S E	89 ± 27^S E	20 ± 11^S E	11 ± 6^S E	26 ± 8^S E
		5000	42 ± 6^S E	84 ± 8^S E	20 ± 3^S E	6 ± 2^S E	27 ± 7^S E
	DAUN	1.00	327 ± 63
	NaN3	2.00		1613 ± 11	918 ± 30
	9-AA	50.0				754 ± 194
	NQO	2.00					1734 ± 65
With Activation	DMSO		44 ± 5	109 ± 11	19 ± 3	9 ± 2	32 ± 5
	Art. 101071	500	48 ± 2^S B	110 ± 20^S B	18 ± 3^S B	7 ± 2^S B	30 ± 1^S B
		889	48 ± 3^S E	124 ± 1^S E	17 ± 10^S E	7 ± 4^S E	30 ± 5^S E
		1580	51 ± 10^S E	135 ± 17^S E	21 ± 6^S E	8 ± 6^S E	32 ± 6^S E
		2810	36 ± 12^S E	116 ± 4^S E	19 ± 4^S E	7 ± 4^S E	31 ± 3^S E
		5000	47 ± 6^S E	116 ± 12^S E	26 ± 5^S E	12 ± 2^S E	35 ± 6^S E
	2-AA	2.00	353 ± 15	591 ± 33
	2-AA	5.00			155 ± 5	111 ± 2
	2-AA	10.0					209 ± 12

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide,S: Plated as suspension, B: Precipitation at beginning of experiment, E: Precipitation until end of experiment

Table 3: Historical Data - Negative Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	402	410	397	413	270	282	276	287	385	398
Number of Values	78	79	77	80	45	47	46	48	74	76
Minimum	23	25	82	101	20	18	23	20	16	17
Maximum	45	59	138	157	34	40	36	43	39	48
Mean	34	40	110	126	27	26	28	30	30	36
Standard Deviation	5.2	7.0	11.7	11.9	3.2	3.9	3.5	4.4	4.9	5.9

Table 4: Historical Data - Positive Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	DAUN	2-AA	NaN3	2-AA	NaN3	2-AA	9-AA	2-AA	NQO	2-AA
Total Plates	201	205	199	207	135	141	138	144	193	199
Number of Values	79	79	77	80	45	47	46	48	74	76
Minimum	117	105	412	512	583	92	122	125	395	112
Maximum	887	1632	2075	3337	1847	390	2882	1103	2286	1313
Mean	352	529	1337	1262	900	200	1175	418	1613	347
Standard Deviation	146.3	296.1	316.8	435.4	193.4	64.2	564.2	201.3	438.7	192.4

Applicant's summary and conclusion

Conclusions:

It can be assumed that the experimental result of the test item is also valid for the anhydrous test item. The test item was considered to be non-mutagenic with and without metabolic activation in bacteria. It can be assumed that the experimental result for the test item is also valid for the anhydrous test item.

Executive summary:

The present study was conducted to investigate the test material for its mutagenic potential in a bacteria! reverse gene mutation assay in the absence and in the presence of a rat liver metabolizing system (S9 mix).

The investigations for the mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with p-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1^st and 20% S9 in the 2^nd series, respectively.

Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for solvent control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following treatment of all the tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.

It can be assumed that the experimental result for the test item is also valid for the anhydrous test item. The test item was considered to be non-mutagenic with and without metabolic activation in bacteria.