ALFERROCK

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No in vitro genotoxicity studies conducted with the target substance are available.

According to the results of the bioelution testing, aluminium has the highest relative release into artificial physiological media representing relevant exposure routes and therefore was considered as main constituent for the human health hazard/risk assessment of the substance (Klawonn, 2021b, see IUCLID section 7.1.1).

In the absence of substance specific data for Alferrock, a conservative approach to hazard evaluation is to assume that aluminium released from Alferrock shows the same systemic hazards as other aluminium compounds with similar or higher bioavailability. Read-across to aluminium compounds with a high release of aluminium ions (soluble Al salts) is considered to represent a worst-case approach that overestimates toxicological properties of the target compound.For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Aluminium is considered the relevant moiety for human health hazard assessment. The genotoxic potential of aluminium is assessed by a weight of evidence approach regarding several in vitro and in vivo studies with various highly soluble aluminium compounds used as read-across source substances. In summary, these studies are considered negative with respect to genotoxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

Remarks:

review article - results of various in vitro and in vivo studies are reported

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Remarks on result:

other: Report is a review article

Various studies cited in a review article.

Conclusions:

Based on the results it can be stated that aluminum compounds are not genotoxic in the absence of cytotoxicity.

Executive summary:

In a review article by Krewski et al., 2007 several in vitro studies regarding the genotoxic potential of aluminum compounds are summarized. Some early studies reported chromosome aberrations upon aluminum chloride exposure, i.e. in spermatocytes of grasshoppers (10 mg/0.21 g bw, Manna and Parida, 1965), in mammalian peritoneal cells (Nashed et al., 1975) and bone marrow cells (0.1 M aluminum chloride 1 mL/30 g bw, acute ip. dose, Manna and Das, 1972). More recently, Roy et al., 1990 reported micronuclei formation and sister chromatic exchange upon 11.6 µmol/mL aluminum chloride in human blood lymphocytes. Stimulation of chromosomal protein crosslinking by aluminum chloride has been reported by Wedrychowski et al., 1986a, and 1986b in ascites hepatoma cells from Sprague-Dawley rats. No crosslinking, in either cytotoxic or non-cytotoxic concentrations of aluminum chloride could be seen in Burkitt human lymphoma cells transformed with an Epstein-Barr virus (Costa et al., 1996).

In bacteria, aluminium compounds have been considered to be non-mutagenic. At concentrations of 1 to 10 mM, Al2O3, AlCl3 and Al2(SO4)3 were negative in the REC-assay with Bacillus subtilis (Kada et al, 1980). The absence of mutagenic effects of aluminium compounds on various bacterial strains including Salmonella typhimurium and Escherichia coli demonstrated in earlier studies has been supported by the findings of more recent studies (Marzin & Phi 1985, Shimizu et al. 1985).

In the study by Oberly et al., 1982 aluminum chloride did not induce forward mutations at the thymidine kinase locus in the mouse lymphoma assay (see IUCLID entry in section 7.6.1 for details).

Importantly, two in vitro studies (Migliore et al, 1999 and Banasik et al., 2004) found genotoxic effects of aluminum compounds only concomitant to cytotoxicity (see IUCLID entries in section 7.6.1 for details).

The cell toxicity of aluminum prior genotoxic effects is supported by the Caicedo et al., 2007 (see IUCLID entry in section 7.6.1 for details) who analyzed the DNA damage provoked by the aluminum metal ion in human Jurkat T-cells. The study used aluminum concentrations of 0.05, 0.1, 0.54, 1 and 5 mM for 48 hours; DNA damage was assessed by single cell electrophoresis (comet assay). Only the highest concentration of 5 mM induced significant DNA damage, concomitant with apoptosis rates exceeding 50 percent. Another comet assay study by Lankoff et al., 2006 used aluminum chloride in doses of 1, 2, 5, 10 and 25 µg/ml exposing human peripheral blood lymphocytes for 72 hours. Olive tail moments increased in association with apoptosis up to a concentration of 10 µg/mL aluminum chloride, while the highest concentration had lower values of the olive tail moment together with an apoptosis rate greater 20 percent.

Based on the results it can be stated that aluminum compounds are not genotoxic in the absence of cytotoxicity and the author stated, that in agreement with their non-carcinogenic activity, aluminum compounds failed to show positive results in most short-term and animal experiments to determine genotoxic potential of aluminum compounds lead to contradictory results with a suggestion of an anti-genotoxic potential.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

bacteria, other: Bacillus subtilis

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 98

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 102

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Conclusions:

Under the described test conditions, the test compound AlCl3 is considered to be negative in the Bacillus subtilis Rec Assay.

Executive summary:

In a Bacillus subtilis Rec Assay, the strain H17 Rec+ (rec+ arg try) and M45 Rec- (rec45 arg try) were exposed to aluminum chloride. Only minor information were presented about the test compound concentration, the solubility and the used solvent/vehicle. Moreover, it is not clear, if postive controls were used during the test . Due to the simplicicity of the assay and the well described protocol of Rec Assay it can nevertheless be stated, that under the decribed test conditions, the test compound aluminum chloride is considered to be negative in the Bacillus subtilis Rec Assay.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to threshold toxic dose (>1000 nM) that decreases bacterial background lawn

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 98

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Conclusions:

Aluminium chloride hexahydrate (at concentrations of 10-1000 nM/plate) was negative in the plate-incorporation assay, conducted similar to the OECD 471, using TA 102 S. typhimurium strain.

Executive summary:

In a reverse gene mutation study bacteria strain TA 102 S. typhimurium was exposed to aluminium chloride hexahydrate in water at concentrations of 10 , 30, 100, 300 and 1000 nM/plate in the absence of mammalian metabolic activation in the plate-incorporation test.

Aluminium chloride hexahydrate was found to be non mutagenic at all tested concentrations.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Al caused a slight decrease in viability of the TA98 bacteria, although this was not statistically significant at the doses tested (for more information, please refer to Table 1 in "Any other information on results incl. tables")

Vehicle controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Species / strain:

S. typhimurium TA 102

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Additional information on results:

Al alone caused a slight decrease in viability of the TA98 bacteria, although this was not statistically significant at the doses tested (Table 1)

Table 1: Viability of TA98 Bacteria Exposed to Al

Treatment (ppm)	Relative percent viability (%) (mean ± SEM)
Control	100.00 ± 8.02
Al (0.5)	107.84 4 5.27
Al (3.0)	90.49 ± 6.45
Al (5.0)	94.69 ± 2.97
Al (7.0)	93.66 ± 5.79

 

Conclusions:

Aluminium chloride (at concentrations of 0.3 and 3.0 mg/L) was negative in the preincubation assay, conducted similar to the OECD 471, using TA98 S. typhimurium strain.

Executive summary:

 In a reverse gene mutation assay study bacteria strain TA98 of S. typhimurium was exposed to Al as AlCl3 in 0.1 M potassium phosphate buffer (PPB, pH 7.4) at concentrations of 0.3 mg/L and 3.0 mg/L (0.3 and 3.0 ppm) in the absence of mammalian metabolic activation in the pre-incubation assay.

There was no evidence of induced mutant colonies over background. Therefore Al at tested concentrations, was found to be non mutagenic as assessed by the preincubation assay using TA98 cells.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

disregarded due to major methodological deficiencies

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Chromosome aberrations and mitotic indices in different cell cycle phases

Cell Cycle Phase	Dose [µM]	MI (%)	relative MI (%)	CAs			Polyp	End
				Gaps	Breaks	Total
G1	Control	5.6	100	1	0	1	0	0
	5	3.4*	61	4	6	10*	6	10*
	10	2.4*	43	8	8	16*	10*	9*
	15	1.9*	34	8	9	17*	9*	9*
	25	1.8*	32	8	14	22*	14*	8*
	DOX	3.1*	55	5	4	9*	2	4
G1/S	Control	5.5*	100	2	0	2	0	1
	5	2.8*	51	12	0	12*	0	0
	10	1.9*	35	15	9	24*	1	0
	15	1.5*	27	14	8	22*	0	1
	25	1.2*	22	18	10	28*	1	0
	DOX	3.2*	58	17	6	23*	5	0
S, 1h	Control	5.4	100	2	0	2	0	0
	5	2.3*	43	28	8	36**	0	0
	10	1.7*	31	35	11	46**	0	0
	15	1.2*	22	30	14	44**	0	0
	25	1.0	19	43	19	62**	0	0
	DOX	2.4*	44	10	7	17*	1	1
S, 6h	Control	4.9	100	2	0	2	0	0
	5	1.4*	29	27	9	36**	0	0
	10	1.0*	20	23	12	35**	0	0
	15	0.5**	10	7	8	15*	0	0
	25	0.2**	4	10	13	23*	0	0
	DOX	1.9*	39	14	10	24*	3	0
G2	Control	5.8	100	8	0	8	0	1
	5	3.5*	60	15	4	19*	10*	2
	10	3.0*	51	17	6	23*	14*	0
	15	2.9*	50	23	8	31*	20*	4
	25	2.4*	41	33	14	47**	27*	5
	DOX	5.2*	90	24	8	32*	7	4

Polyp: polyploid cells; End: endoreduplication; DOX: Doxorubicin

* P<0.05; **P<0.01

Conclusions:

Based on the deviations in data collection, analysis and reporting, the study results are considered ambiguous.

Executive summary:

In a mammalian cell cytogenetic assay peripheral human lymphocyte cultures were exposed to aluminum chloride, solved in methanol, at concentrations of 5, 10, 15 and 25 µM.

To investigate the potential of aluminum chloride to induce chromosome aberrations during various stages of the cell cycle, treatment schedules were adapted to G1 (52 h), transition phase G1-S (starting 24 hours after phytohaemagglutinin stimulation) and S phase (1 or 6 hours, starting 24 hours after phytohaemagglutinin stimulation) of the cells. The treatment with aluminum chloride led to cytotoxicity, determined by the mitotic index. In accordance with OECD guideline 473 a reduction of the mitotic index to 45 +/- 5% of the concurrent negative control is acceptable. Moreover, at least three testing concentrations should meet the criteria (e.g. appropriate cytotoxicity). The only experiment, which meets the criteria was the G2 cell cycle analysis. All treatments lead to a statistically significant increase of chromosomal aberration in comparison to the untreated control. The positive results could be atrributed to cytotoxicity as all test concentrations induce cytotoxicity in the higher end of the cytotoxicity range as recommended in the OECD test guideline. The positive control for the chromosome aberration test (doxorubicine) induced the appropriate response.

It has to be noted, that in this study the recorded gaps were included in total aberration frequency. The OECD guideline 473 recommended not to include gaps in the total aberration frequency. Thus, a statistical analysis does not include gaps. In summary, based on the deviations in data collection, analysis and reporting the study results are considered ambiguous and no clear conclusion may be drawn from this study .

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

disregarded due to major methodological deficiencies

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: peripheral-blood lymphocytes from 3 male adults aged between 20-30 years

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
No

TEST-SPECIFIC CONFOUNDING FACTORS
No data presented

RANGE-FINDING/SCREENING STUDIES:
No data presented

COMPARISON WITH HISTORICAL CONTROL DATA:
No

In the three treatment groups, AlCl₃ induced both micronuclei and apoptosis. At the highest dose of AlCl₃the frequency of micronuclei declined (0-4 h and 0-72 h) or remained similar for the highest concentration levels (10 -72 h) in comparison to the dose group below. According to the authors, this effect, which was especially strong in cells treated during the G₀/G₁ phase (0 -4 h) and the whole cell cycle (0 -72 h), can be accounted for by apoptotic cell death. The highest frequency of apoptosis was observed in cells treated either during G₀/G₁ or the whole cell cycle (80% apoptosis). The frequencies of micronuclei in cells treated during G₀/G₁ or S/G₂ (10 -72 h) were comparable. In contrast, the frequency of apoptosis in cells treated during the S/G₂ phase remained below 40% for all concentrations, while the level increased to 80% at the highest concentration level in cells treated during the G₀/G₁ phase and the whole cell cycle. The negative control showed an apoptosis level of approximately 15% and less than 20 micronuclei per 1000 binucleated lymphocytes.

Conclusions:

The frequency of micronucleated cells was dose-related increased and parallel to the induction of apoptosis after treatment with aluminum chloride. As no positive control were included in the experiment, the validity of the test is questionable.

Executive summary:

In a mammalian cell micronucleus test similar to the OECD test guideline 487 primary human peripheral-blood lymphocytes cultures were exposed in vitro to aluminum chloride at concentrations of 0, 1, 2, 5, 10 and 25 µg/mL without metabolic activation for 0-4, 10-72 or 0-72 h. A cytotoxicity parameter as recommended by OECD guideline 473 was not assessed. Instead, apoptosis levels were determined and used for cytotoxicity assessment. The apoptosis levels were determined with up to 40 % , except the 25 µg/mL dose group, which induced roughly 80% apoptosis after 0-4h and 0-72h treatment to aluminum chloride. Micronuclei frequency showed an parallel increase with the levels of apoptosis with one exception, at 80% observed apoptosis in the 25 µg/mL dose group after 0-4h and 0-72h treatment the micronuclei values dropped by almost 50% in comparison to the dose group below (10 mg/mL, 40% apoptosis). The results were not considered for the evaluation of the mutagenic potential of aluminum chloride. No positive controls were reported, thus the validity of the test was not verified.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: Human Jurkat T-lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

TABLE 1: Concentrations of Metals Required to Induce a a Significant Harmful Effect (DNA Damage, Apoptosis, Viability, and Proliferation Inhibition) on T-Helper Jurkat Cells
Metal Concentrations (mM)	DNA Damage	Apoptosis	Viability	Proliferation Inhibition	Average Concentration of Four Parameters
V	0.05	0.05	1.0	0.05	0.29
Ni	0.05	0.1	5.0	0.5	1.41
Co	5.0	5.00	0.5	0.1	2.65
Cu	>5	0.5	5.0	0.1	>2.65
Nb	>5	0.5	0.5	>5	>2.75
Mo	>5	1.0	>5	0.5	>2.87
Zr	5.0	0.5	5.0	>5	>3.875
Be	>5	5.0	1.0	5.0	>4
Cr	>5	>5	>5	>5	>5
Al	>5	5.0	>5	>5	>5
Fe	>5	5.0	>5	>5	>5
Significant effect:
DNA damage: IDD>75.
Apoptosis:>50% caspase 9-positive cells.
Viability: >50% PI-positive cells.
Proliferation inhibition: p<0.05 significance in metal-treated cells CPMs reduction compared to untreated controls.

Conclusions:

Aluminum chloride solution is considered to be non-genotoxic in a Comet assay.

Executive summary:

In an in vitro Comet assay, human Jurkat T-lymphocyte cells cultured in vitro were exposed to aluminum chloride solution at concentrations of 0.05, 0.1, 0.5, 1.0 and 5 mM in the absence of mammalian metabolic acivation.

For assessment of cytotoxicity, apoptosis, cell proliferation and cell viability were measured. Aluminum chloride induced >50% caspase-9 positive cells at 5 mM concentration. Aluminum chloride solution showed no effects on cell proliferation and cell viability at the concentrations tested. No significant increase of DNA damage, measured by using an index of DNA damage (IDD, average tail lenghth of 50 cells) were observed.

Therefore, Aluminum chloride solution is not genotoxic in the Comet assay.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: after adding the chemical to the medium, a dramatic change in pH occured over the 4h expression period

Remarks on result:

other: all strains/cell types tested

Table1: Mutagenic response after treatment with AlCl3

Chemical	Dose [µg/mL]	Percent total survival	Mutation frequency	Increase over solvent (-fold)
Solvent	0	100	2.5	-
EMS	620	31	85.0	34.0
AlCl3	625	63	5.6	2.2
	600	38	5.0	2.0
	590	42	4.9	2.0
	580	88	4.3	1.7
	570	69	5.0	2.0

Conclusions:

The test substance AlCl3 did not induce gene mutation in the TK locus in L5178Y mouse lymphoma cells under the tested experimental conditions.

Executive summary:

In a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells cultured in vitro were exposed to AlCl3, solved in water, at concentrations of 570, 580, 590, 600, and 625 µg/mL in the absence of mammalian metabolic activation. The induced mutation frequencies without metabolic activation were not increased by more than 2 fold in comparison to the untreated control, except the 625 µg/mL dose group dose with a 2.2 fold increase. The results of the mutant frequencies did not fulfill the criteria for a positive study result outcome. The positive control ethyl methanesulfonate (EMS) did induce the appropriate response.

In summary, the test item AlCl3 did not induce gene mutation in L51178Y mouse lymphoma cells.

This study is classified as acceptable and satisfies the requirement for test guideline OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 9

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

other: The results obtained are considered not biologically relevant.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

N.A.

Table 1: Donor A_ Results

Chemical	Dose [µM]	BN scored	MN BN^1	MN BN/1000 +/-SD	BN (%)^1
negative control	0	2000	12	6.0 +/- 1.4	33.0
Griseofulvin	43	2000	131	65.5 +/- 34.6***	22.7
Mitomycin C	0.51	2000	121	60.5 +/- 12.0***	22.5
Al2(SO4)3	500	2000	14	7.0 +/- 2.8	21.2
	1000	2000	23	11.5 +/- 2.1*	25.0
	2000	2000	30	15.0 +/-1.4**	27.5
	4000	2000	11	5.5 +/- 0.7	29.5

Table 2: Donor B_Results

Chemical	Dose [µM]	BN scored	MN BN^1	MN BN/1000 +/-SD	BN (%)^1
negative control	0	2000	8	4.0 +/- 1.4	45.6
Griseofulvin	43	1555	46	29.4 +/- 0.8***	18.9
Mitomycin C	0.51	2000	209	104.5+/- 7.8***	17.9
Al2(SO4)3	500	2000	19	9.5 +/- 3.5*	47.2
	1000	2000	28	14.0 +/-0.0***	46.7
	2000	2027	20	9.9 +/-3.0*	53.2
	4000	2000	18	9.0 +/- 0.0*	33.9

1: MN BN: number micronucleated binucleated cells, BN (%): binucleated cells/total cells

* Fishers exact test: *P<0.05, **P< 0.01, ***P<0.001

Conclusions:

The results obtained in the in vitro mammalian cell micronucleus test after treatment with aluminum sulfate are considered not biologically relevant. In certain dose groups a slight increase in micronuclei frequency was observed in comparison to the untreated control, but no dose relationship was observed.

Executive summary:

In a mammalian cell micronucleus test conducted similar to OECD guideline 487, human lymphocytes were exposed in vitro to aluminum sulfate at concentrations of 0, 500, 1000, 2000 and 4000 µM without metabolic activation for 48 hours.

Micronuclei frequency was counted in 2000 cells for each concentration of each donor, showing a slight increase in micronucleus frequency. This increase is considered not biologically relevant, as in both donor blood experiments no dose-relationship was observed (the micronucleus frequency of the highest dose tested in donor 1 was even below the negative control values). The positive controls did induce distinct and biologically relevant increases of the micronucleus frequency. Under the test conditions, aluminum sulfate induced micronuclei without a dose relationship in human lymphocytes.The results show a certain degree of variability between the two donors and question the relevance of the slight increase of micronuclei frequency. Moreover, the dose levels used induced no apparent toxicity (change in frequency of binucleate cells) and no relationship between toxicity, dose and the level of micronucleated cells was observed.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity studies conducted with the target substance are available.

According to the results of the bioelution testing, aluminium has the highest relative release into artificial physiological media representing relevant exposure routes and therefore was considered as main constituent for the human health hazard/risk assessment of the substance (Klawonn, 2021b, see IUCLID section 7.1.1).

In the absence of substance specific data for Alferrock, a conservative approach to hazard evaluation is to assume that aluminium released from Alferrock shows the same systemic hazards as other aluminium compounds with similar or higher bioavailability. Read-across to aluminium compounds with a high release of aluminium ions (soluble Al salts) is considered to represent a worst-case approach that overestimates toxicological properties of the target compound. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

In a review article by Krewski et al., 2007 several in vivo studies investigating the genotoxic potential of aluminum compounds are summarized. In summary of the reported studies using various aluminum compounds, the author stated, that in agreement with their non-carcinogenic activity, aluminum compounds failed to show positive results in most short-term assays and animal experiments to determine genotoxic potential of aluminum compounds lead to contradictory results with a suggestion of an anti-genotoxic potential.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

review article - results of various in vitro and in vivo studies are reported

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Various studies cited in a review article.

Conclusions:

Based on the results evaluated in a weight-of-evidence approach it can be stated that aluminium compounds are not genotoxic in the absence of cytotoxicity.

Executive summary:

In a review article by Krewski et al., 2007 several in vivo studies regarding the genotoxic potential of aluminum compounds are summarized. For aluminum chloride and aluminum sulfate the results of 2 studies each depicts a similar picture as the in vitro tests, with observed genotoxicity upon high dosage levels, but not upon lower doses. The 2 studies with aluminum sulfate were performed by Roy et al., 1992 and Dhir et al., 1993 using between 100-500 mg/kg bw. The author reported an increase in micronucleated polychromatic peripheral erythrocytes 24 hours after a second aluminum dose of 500 mg/kg bw but not upon 250 mg/kg bw. Dhir et al., 1993 reported sister chromatid exchange, detected by bromodeoxyuridine, 24 hours after injection in male Swiss albino mice. For aluminum chloride, positive findings were reported in a preliminary study by Manna and Das, 1972 using 10-100 mmol/mouse i.p. causing chromosome aberrations. In contrast, Spotheim-Maurizot et al., 1992 did not observe genotoxic effects of aluminum chloride upon doses of 0.2 mM.

A short term study using aluminum nitrilotriacetate complex administered i.p. at a dosage of 7 mg/Al/kg caused no changes in the formation of 8-hydroxydeoxyguanosie within 24-48 hours (Umemura et al., 1990). A 1.2% aluminum clofibrate content in the diet of F-344 rats raised within 1 -12 month the hepatic peroxisomal beta-oxidation enzyme activity. Such effects are known for clofibrate, and therefore are not related to aluminum (Takagi et al., 1990).

Some other positive findings are summarized in the review of Krewski et al., 2007 in which complex mixtures that entailed aluminum were analyzed. These positive findings may, however, rather relate to the non-aluminum fractions of the mixtures. In the study by Bauer et al., 1995 vacuum pump oils contaminated with waste products from a BC13/C12 aluminum plasma etching process were given to female Wistar rats and the approximated aluminum concentration was only 2000 ppb. Another mixture study was performed by Sivikova and Dianovsky, 1995 administering ionic forms of metals from an aluminum refining plant in distilled water for one year to sheep. The total aluminum concentration delivered was 1.1 or 2.4 mmol/Al/animal/day. Only the higher dose resulted in an increase of sister chromatid exchanges in the cultures lymphocytes.

Thus, in summary of the reported studies using various aluminum compounds, the author stated, that in agreement with their non-carcinogenic activity, aluminum compounds failed to show positive results in most short-term and animal experiments to determine genotoxic potential of aluminum compounds lead to contradictory results with a suggestion of an anti-genotoxic potential.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

 No substance specific genotoxicity studies are available for target substance.

According to the results of the bioelution testing, aluminium has the highest relative release into artificial physiological media representing relevant exposure routes and therefore was considered as main constituent for the human health hazard/risk assessment of the substance (Klawonn, 2021b, see IUCLID section 7.1.1).

In the absence of substance specific data for Alferrock, a conservative approach to hazard evaluation is to assume that aluminium released from Alferrock shows the same systemic hazards as other aluminium compounds with similar or higher bioavailability. Read-across to aluminium compounds with a high release of aluminium ions (soluble Al salts) is considered to represent a worst-case approach that overestimates toxicological properties of the target compound. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Aluminium chloride was tested negative in the Bacillus subtilis Rec mutagenicity screening assay. This assay is detecting DNA damage, which is subjected to cellular recombination repair (Kada et al., 1980). Caicedo et al., (2007) reported, that aluminium chloride was tested negative in an in vitro Comet assay. Oberly et al., 1982 reported negative results in a L5178Y mouse lymphoma mutagenicity assay (OECD 476) using aluminium chloride. Two in vitro studies (Banasik et al., 2005 (in vitro MNT), Lima et al., 2007 (in vitro CA)) found genotoxic effects of aluminium chloride only concomitant to severe cytotoxicity. The positive effect is not considered relevant due to the increased cytotoxicity, disqualifying a distinction of genotoxic from cytotoxic events.

In a mammalian cell micronucleus test conducted similar to OECD guideline 487, human lymphocytes were exposed in vitro to aluminium sulfate at concentrations of 0, 500, 1000, 2000 and 4000 µM without metabolic activation for 48 hours. The results revealed a slight increase in micronucleus frequency. As in both blood donor experiments no dose-relationship was observed (the micronucleus frequency of the highest dose tested in donor 1 was even below the negative control values). Overall, the results are considered ambiguous due to the before mentioned reasons.

Supporting data was received by the review article by Krewski et al., 2007. In a weight of evidence evaluation of the data presented by Krewski, it can be stated, that aluminium compounds are not genotoxic in the absence of cytotoxicity.

 

Additionally in a review article by Krewski et al., 2007 several in vivo studies regarding the genotoxic potential of aluminum compounds are summarized.In summary of the reported studies using various aluminum compounds, the author stated, that in agreement with their non-carcinogenic activity, aluminum compounds failed to show positive results in most short-term assays and animal experiments to determine genotoxic potential of aluminum compounds lead to contradictory results with a suggestion of an anti-genotoxic potential. In summary of the available data from various read-across partners, no genotoxic potential at non-cytotoxic concentrations was observed. Therefore, it can be concluded that aluminium released from Alferrock can be considered as not genotoxic.

This is in the line with conclusions made by different regulatory bodies (EFSA, SCCS) stating that “metallic aluminium, its oxides, and common aluminium salts have not been shown to be either genotoxic or carcinogenic” as reported in the document “Final Opinion on tolerable intake of aluminium with regards to adapting the migration limits for aluminium in toys” (European Union, 2017). In addition, the European Food Safety Authority concluded in its Scientific Opinion on the Safety of aluminium from dietary intake (The EFSA Journal (2008) 754, 1-34) that genotoxicity is unlikely to be of relevance for humans exposed to aluminium via the diet.

Justification for classification or non-classification

Based on data from soluble aluminium compounds, aluminium released from Alferrock is considered negative regarding genetic toxicity. Therefore, classification according to the criteria set out in Annex I of Regulation (EC) 1272/2008 (CLP criteria) is not warranted