Bis(acetato-O)hydroxyaluminium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria (reference 7.6.1 - 1).

The test substance did not induce any chromosome aberrations over background (reference 7.6.1 - 2).

The test substance did not induce gene mutations at the HPRT locus in V79 cells (reference 7.6.1 - 3).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

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

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.

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.

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-09-24 to 2018-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test) (migrated information)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: female, approximately 18 to 35 years of age (non-smoker), 2
- Whether whole blood or separated lymphocytes were used: whole blood
- Mitogen used for lymphocytes:

MEDIA USED
The pre-treatment of blood with Phytohemagglutinin (PHA) E was done under cooled conditions (1.5 hours incubation). Mitotic stimulation of lymphocytes in chromosome medium B (ready-to-use, purchased from Biochrom) was done for 48 hours at 37°C/ 5% CO2).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

62.5, 125, 250 and 500 µg/mL (-S9 mix, 5 hours exposure)
7.81, 15.6 and 31.3 µg/mL (-S9 mix, 29 hours exposure)
125, 250, 500 and 1000 µg/mL (+S9 mix, 5 hours exposure)

Cytotoxicity was the determining factor for the top dose selection.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Preliminary experiments to evaluate the solubility of the test item in various solvents showed that DMSO was the only suitable one. Due to limited solubility, the test item was applied as homogenous suspension. The final concentration of DMSO present in cell culture medium was 1.0%.

- Justification for percentage of solvent in the final culture medium: Analysis of the historical data of the laboratory and experience of other research groups showed that such amounts of the selected solvent has no influence on the mutation frequency in this test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

CPA

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

MMC

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of slides per concentration: 2 (4 for solvent control)
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT SCHEDULE:
- Exposure duration/duration of treatment: 5 and 29 hours (-S9 mix); 5 hours (+S9 mix)


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: 74 hours after initiation of the cultures, colchicine was added (to yield a final concentration of 0.1 pg/mL) to arrest lymphocytes in mitosis. Then, cultures were incubated for another 3 hours.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): On the day of preparation, the cell cultures were placed into centrifugation tubes and spun for 10 min at 169xg. The culture medium was replaced by KG solution (0.2%) for hypotonic treatment. For fixation of cells, the tubes were placed into an ice bath and fixative (methanol :acetic acid, 3:1) was added. Cultures were incubated for 20 min at room temperature, then spun for 10 min/169xg. Two further fixation/centrifugation cycles followed. After the third fixation cycle, the supernatant was aspirated and the cell pellet carefully resuspended. From each parallel culture, two slides were prepared. The cell suspension was dropped onto wet, pre-cooled glass slides. The slides were dried for at least three days, then stained in aceto-orcein, immersed in xylene and made permanent with Entellan®.
- Number of metaphases analysed per slide: 150 for structural aberrations
- Determination of polyploidy: Yes, in case it was seen
- Determination of endoreplication: Yes, in case it was seen

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: The determination of the mitotic index was based on scoring 1000 cells per slide.

OTHER:
Criteria for Study Acceptability
• The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database
• Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control
• Cell proliferation criteria in the solvent control should be fulfilled
• All three experimental conditions were tested unless one resulted in positive results
• Adequate number of cells and concentrations are analyzable
• The criteria for the selection of top concentration are fulfilled

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test, or occurs across at least two concentrations
c) any of the results is outside the distribution of the historical negative control data (e.g. 95% control limits).
When all of these criteria are met, the test item is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. 95% control limits).
The test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For further statistical analysis, the numbers of aberrant metaphases (gaps excluded) were used. Pairwise comparisons within each experiment were performed. Each treatment group was compared to the concurrent negative control. For these comparisons, the Fisher's Exact Test was performed against one-sided alternatives. To control the global a-level (a = 0.05), a correction for multiple testing was applied (Bonferroni-Holm). The p-values of these comparisons are presented in the table's section. Statistical significances observed in cases where the mean proportion of aberrant metaphases in a treated group is lower compared to the concurrent negative control group are generally considered not relevant for the test hypothesis. To investigate a potential concentration-dependent effect, the Cochran-Armitage trend test was applied.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 1000 µg/mL after 5 hours exposure and was more pronounced in cultures continuously exposed for 29 hours (> 62.5 µg/mL) in the absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Change of pH did not occur.
- Data on osmolality: Change of osmotic value did not occur.
- Precipitation and time of the determination: Precipitation was observed at concentrations >= 1000 µg/mL (5 hours exposure) and >= 500 µg/mL (29 hours exposure)

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The mean values of aberrant metaphases (gaps excluded) in the negative controls of all experiments ranged from 1.33% to 2.67%. The mitotic index of the negative controls was within the historical control data indicating sufficient cell proliferation. A clear increase of aberrant metaphases was found for the positive controls, i.e. MMC and CPA (please refer to table 1 "Any other information on results incl. tables"). Statistical analysis in all cases yielded significant results. An appropriate number of cells and concentrations were analyzable. The experiments were therefore accepted as valid.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: A clear reduction of the mitotic index (25% of the concurrent solvent control) was induced by the test item at 2000 µg/mL in the presence of metabolic activation (S9 mix) after 5 hours exposure, thus limiting the top dose for evaluation to 1000 µg/mL. In cultures exposed for 5 or 29 hours in the absence of S9 mix, multiple signs of cytotoxicity, i.e. reduced cell density, morphological changes of cells and chromosomes were observed at concentrations above 500 µg/mL or 31.3 µg/mL, thus limiting the appropriate top dose.

- Genotoxicity results
In none of the dose groups treated with the test item, the mean percentage of aberrant metaphases (excluding gaps) significantly exceeded the percentages of the concurrent negative controls. A trend test applied was not significant, giving evidence for the absence of any concentration-related effects. No relevant treatment-related increase of polyploid cells or endoreduplications was observed.

HISTORICAL CONTROL DATA:
MMC (range, mean±SD, 95 % CI): 4.5 - 30 %, 12.4 ± 4.8 %, 2.7 - 22.1 % (data from 42 experiments)
CPE (range, mean±SD, 95 % CI): 6.5 - 32.0 %, 15.4 ± 5.5 %, 4.4 - 26.4 % (data from 34 experiments)
- Negative (solvent/vehicle) historical control data for mean aberrant metaphases (excl. gaps):
without S9 mix (range, mean±SD, 95 % CI): 0.0 - 4.8 %, 2.2 ± 1.1 %, 0.0 - 4.3 % (data from 46 experiments)
with S9 mix (range, mean±SD, 95 % CI): 0.3 - 5.8 %, 2.8 ± 1.5 %, -0.2 - 5.9 % (data from 40 experiments)

Table 1 results

Treatment group	Concentration [µg/mL]		Rel. Mitotic Index [%]^a	Polyploid metaphases^b	Endomitotic metaphases^b	Aberrant metaphases [%]
						incl. Gaps	excl. Gaps	exchanges
Without S9 mix
exposure: 5 hours
Solvent control	0.0	100.0		2	0	3.17	2.67	0.00
test substance	62.5	88.4		0	0	3.67	2.67	0.00
	125	75.5		1	0	3.33	3.00	0.33
	250	75.9		0	0	5.00	4.33	0.00
	500	77.3		0	0	3.00	2.33	0.00
MMC	0.5	88.0		0	0	19.7	19.3*	6.67
exposure: 29 hours
Solvent control	0.0	100.0		2	0	1.67	1.33	0.00
test substance	7.8	96.7		2	0	1.33	1.00	0.00
	15.6	110.0		3	0	2.67	2.67	0.00
	31.3	127.0		2	0	2.00	1.67	0.00
MMC	0.2	91.4		1	0	16.0	16*	3.00
with S9 mix
exposure: 5 hours
Solvent control	0.0	100.0		2	0	1.33	1.33	0.00
test substance	125	78.8		1	0	2.33	2.33	0.00
	250	81.1		0	0	1.33	1.33	0.00
	500	69.8		0	0	2.67	2.67	0.00
	1000^PE	69.8		0	0	1.00	0.67	0.00
CPE	2.3	80.2		0	0	14.0	14*	3.33

a: % of solvent control, b: recorded during evaluation

MMC: Mitomycin C, CPA: Cyclophosphamide

Statistical significance: *: statistically significant (Fisher's Exact Test)

PE: Precipitation at the end of exposure

Conclusions:

Treatment of cultured human peripheral blood lymphocytes with the test substance (Aluminium hydroxide diacetate monohydrate) did not relevantly increase the proportion of cells with aberrant chromosomes under the experimental conditions. Thus, Art. 101071 (Aluminium hydroxide diacetate monohydrate) was not clastogenic in this in vitro test system under the conditions described in the present study. It can be assumed that the experimental result of the test item is also valid for the anhydrous test item.

Executive summary:

The aim of the present study was to investigate the potential of the test item to induce structural chromosomal damage in cultured human peripheral blood lymphocytes. Aluminium hydroxide diacetate monohydrate was investigated in two independent experiments for induction of chromosomal aberrations in human peripheral blood lymphocytes in vitro in accordance with OECD guideline 473. This also included examinations on whether or not the test item may have the potential to induce numerical aberrations, i.e. an increase in endo-reduplications or polyploidy.

The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Phenobarbital/p-Naphthoflavone):

No. of slides evaluated per concentration: Solvent control: Others:	4 2
No. of metaphases evaluated per slide:	150 for structural aberrations
Preparation times (after initiation of the cell cultures: - S9 mix: +S9 mix:	77 hours 77 hours
Exposure periods: - S9 mix: +S9 mix:	5 and 29 hours 5 hours
Solvent for the test item:	Dimethylsulfoxide (DMSO), 1.0% final concentration
Concentrations evaluated: Test item - S9 mix, 5 h: + S9 mix, 5h: -S9 mix, 29 h: Positive controls  - S9 mix: +S9 mix:	62.5; 125, 250 and 500 µg/mL 125, 250, 500 and 1000 µg/mL 7.81, 15.6 and 31.3 µg/mL   0.225 and 0.500 µg Mitomycin C /mL 2.25 µg Cyclophosphamide (CPA) /mL

 

The mean values of aberrant metaphases (gaps excluded) in the negative controls ranged were within the range of negative control values reported for our laboratory. The positive controls. Mitomycin C (MMC) and Cyclophosphamide (CPA), induced the expected clear increase in the proportion of cells with chromosomal aberrations. For the selection of appropriate concentrations for evaluation, cytotoxicity was the determining factor. Cytotoxicity was observed as reduced cell density, morphological changes of cell and chromosomal structure or reduced mitotic activity. No relevant increase in the numbers of chromosomal aberrations, endomitotic or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected. In conclusion, treatment of cultured human peripheral blood lymphocytes with the test substance did not relevantly increase the proportion of cells with aberrant chromosomes under the experimental conditions. Thus, aluminium hydroxide diacetate monohydrate was not clastogenic in this in vitro test system under the conditions described in the present study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-05 to 2018-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes) (migrated information)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

For cell lines:
- Absence of Mycoplasma contamination: It was screened in the master cell stock.
- Doubling time: 12 - 16 h in stock cultures
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Thawed stock cultures were propagated at 37 °C in 75 cm² plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

4 hours (without S9 mix)
0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50, 100 µg/mL

4 hours (with S9 mix)
3.1, 6.3, 12.5, 25, 50, 100 µg/mL

The cultures at 0.8 μg/mL without metabolic activation, and at 0.8 and 1.6 μg/mL with metabolic activation were not continued as a minimum of only four concentrations is required by the guidelines. The cultures at 50.0 and 100.0 μg/mL without metabolic activation, and at 100.0 μg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

- Justification for percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium was 0.5 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

EMS

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

DMBA

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2×10^7
- Test substance added in medium

TREATMENT SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time: 6 days
- Selection time: 9 days
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used, indicate its identity, its concentration: 6-thioguanine, 11 µg/mL

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together. A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

observed at 25 µg/mL (-); 50 µg/mL (+)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant shift of pH.
- Data on osmolality: No relevant shift of pH.
- Precipitation and time of the determination: Precipitation occurred at 55.6 μg/mL and above in the presence and absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 1.7 μg/mL and 500 μg/mL were used. The highest concentration was chosen based on the solubility properties of the test item.
No relevant cytotoxic effect, indicated by a relative cloning efficiency of approx. 50% or below was observed up to 55.6 μg/mL with and without metabolic activation. The cell number of the next two higher concentrations were not counted, since the lowest counted concentration which showed precipitation was 55.6 μg/mL. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 55.6 μg/mL and above in the presence and absence of metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

STUDY RESULTS
No relevant cytotoxic effect indicated by the mean adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation. No relevant increase in the mean mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical laboratory solvent control data. The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups. The mean mutant frequency obtained for the solvent controls in the main experiment was 11.7 mutants per 10^6 cells in the absence of metabolic activation, and 9.9 mutants per 10^6 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 7.0 up to 13.2 mutants per 10^6 cells. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

HISTORICAL CONTROL DATA:
EMS (range, mean±SD, 95 % CI): 53.9 - 872.3 µg/mL, 221.1 ± 102.6 µg/mL, - µg/mL (data from 168 experiments; 150 and 300 µg/mL)
DMBA (range, mean±SD, 95 % CI): 55.6 - 739.9 µg/mL, 191.4 ± 100 µg/mL, - µg/mL (data from 162 experiments; 1.1 and 2.3 µg/mL)
- Negative (solvent/vehicle) historical control data:
without S9 mix (range, mean±SD, 95 % CI): 3.4 - 41.0 µg/mL, 16.9 ± 7.1 µg/mL, 2.7 - 31.2 µg/mL (data from 168 experiments)
with S9 mix (range, mean±SD, 95 % CI): 2.4 - 40.4 µg/mL, 16.9 ± 7.0 µg/mL, 2.9 - 30.9 µg/mL (data from 162 experiments)

Table 1 results

Treatment group	Concentration [µg/mL]	Prec.	S9 mix	relative cloning efficiency I [%]	relative cell density [%]	relative adjusted cloning efficiency I [%]	mutant colonies/10^6 cells	95 % CI
Main Experiment / 4 h treatment                           mean values of culture I and II
Solvent control with DMSO			-	100.0	100.0	100.0	11.7	2.7-31.2
Positive control (EMS)	300.0		-	80.1	92.8	74.3	173.8	2.7-31.2
Test item	0.8		-	100.4	97.1	98.0	#
Test item	1.6		-	97.8	94.7	91.6	8.1	2.7-31.2
Test item	3.1		-	96.9	93.1	90.1	8.2	2.7-31.2
Test item	6.3		-	92.5	83.4	77.5	8.5	2.7-31.2
Test item	12.5		-	87.7	85.4	74.9	8.2	2.7-31.2
Test item	25.0	P	-	70.5	75.7	53.9	7.0	2.7-31.2
Test item	50.0	P	-	##
Test item	100.0	P	-	##
Solvent control with DMSO			+	100.0	100.0	100.0	9.9	2.9-30.9
Positive control (EMS)	300.0		+	97.9	101.5	99.3	103.0	2.9-30.9
Test item	0.8		+	97.6	115.4	112.6	#
Test item	1.6		+	99.1	93.4	92.4	#
Test item	3.1		+	100.5	103.1	103.6	13.2	2.9-30.9
Test item	6.3		+	96.0	102.9	98.7	7.4	2.9-30.9
Test item	12.5		+	102.2	99.9	101.9	9.2	2.9-30.9
Test item	25.0		+	93.9	96.1	90.2	9.6	2.9-30.9
Test item	50.0	P	+	94.5	100.7	95.7	8.0	2.9-30.9
Test item	100.0	P	+	##

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Aluminium hydroxide diacetate hydrate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in accordance with OECD guideline 476. V79 cells cultured in vitro were exposed to the test substance at concentrations of 0, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50 and 100 µg/mL in the presence and absence of metabolic activation (S9 mix). The cultures at 0.8 μg/mL without metabolic activation, and at 0.8 and 1.6 μg/mL with metabolic activation were not continued as a minimum of only four concentrations is required by the guidelines. The cultures at 50.0 and 100.0 μg/mL without metabolic activation, and at 100.0 μg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations.The treatment period was 4 hours with and without S9 mix.

No relevant increase in the mean mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. No relevant cytotoxic effects indicated by the mean adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation. The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical laboratory solvent control data. The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups. The mean mutant frequency obtained for the solvent controls in the main experiment was 11.7 mutants per 10^6 cells in the absence of metabolic activation, and 9.9 mutants per 10^6 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 7.0 up to 13.2 mutants per 10^6 cells. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, aluminium hydroxide diacetate hydrate is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation Test

The present study was conducted to investigate the test material for its mutagenic potential in a bacterial 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 1st and 20% S9 in the 2nd 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.

Chromosome Aberration Assay

The aim of the present study was to investigate the potential of the test item to induce structural chromosomal damage in cultured human peripheral blood lymphocytes. Aluminium hydroxide diacetate monohydrate was investigated in two independent experiments for induction of chromosomal aberrations in human peripheral blood lymphocytes in vitro in accordance with OECD guideline 473. This also included examinations on whether or not the test item may have the potential to induce numerical aberrations, i.e. an increase in endo-reduplications or polyploidy. The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Phenobarbital/p-Naphthoflavone):

No. of slides evaluated per concentration: Solvent control: Others:	4 2
No. of metaphases evaluated per slide:	150 for structural aberrations
Preparation times (after initiation of the cell cultures: - S9 mix: +S9 mix:	77 hours 77 hours
Exposure periods: - S9 mix: +S9 mix:	5 and 29 hours 5 hours
Solvent for the test item:	Dimethylsulfoxide (DMSO), 1.0% final concentration
Concentrations evaluated: Test item - S9 mix, 5 h: + S9 mix, 5h: -S9 mix, 29 h: Positive controls  - S9 mix: +S9 mix:	62.5; 125, 250 and 500 µg/mL 125, 250, 500 and 1000 µg/mL 7.81, 15.6 and 31.3 µg/mL   0.225 and 0.500 µg Mitomycin C /mL 2.25 µg Cyclophosphamide (CPA) /mL

 

The mean values of aberrant metaphases (gaps excluded) in the negative controls ranged were within the range of negative control values reported for our laboratory. The positive controls. Mitomycin C (MMC) and Cyclophosphamide (CPA), induced the expected clear increase in the proportion of cells with chromosomal aberrations. For the selection of appropriate concentrations for evaluation, cytotoxicity was the determining factor. Cytotoxicity was observed as reduced cell density, morphological changes of cell and chromosomal structure or reduced mitotic activity. No relevant increase in the numbers of chromosomal aberrations, endomitotic or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected. In conclusion, treatment of cultured human peripheral blood lymphocytes with aluminium hydroxide diacetate monohydrate did not relevantly increase the proportion of cells with aberrant chromosomes under the experimental conditions. Thus, aluminium hydroxide diacetate monohydrate was not clastogenic in this in vitro test system under the conditions described in the present study.

Mammalian cell gene mutation

The study was performed to investigate the potential of aluminium hydroxide diacetate hydrate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in accordance with OECD guideline 476. V79 cells cultured in vitro were exposed to the test substance at concentrations of 0, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50 and 100 µg/mL in the presence and absence of metabolic activation (S9 mix). The cultures at 0.8 μg/mL without metabolic activation, and at 0.8 and 1.6 μg/mL with metabolic activation were not continued as a minimum of only four concentrations is required by the guidelines. The cultures at 50.0 and 100.0 μg/mL without metabolic activation, and at 100.0 μg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations. The treatment period was 4 hours with and without S9 mix.

No relevant increase in the mean mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. No relevant cytotoxic effects indicated by the mean adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation. The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical laboratory solvent control data. The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups. The mean mutant frequency obtained for the solvent controls in the main experiment was 11.7 mutants per 10^6 cells in the absence of metabolic activation, and 9.9 mutants per 10^6 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 7.0 up to 13.2 mutants per 10^6 cells. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, aluminium hydroxide diacetate hydrate is considered to be non-mutagenic in this HPRT assay.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The results indicate that the substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EC) No 2019/521.