Aluminium sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are conclusive but not suffcient data for the classification of substance Aluminium sulphate with regard to mutagenicity/genetic toxicity.

Link to relevant study records

Reference

Endpoint:

in vitro transformation study in mammalian cells

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restrictions. Well-presented study, with relevant measurement of chemical concentrations

Qualifier:

according to guideline

Guideline:

EU Method B.21 (In Vitro Mammalian Cell Transformation Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Assay:
Test Category: MISCELLANEOUS CATEGORY
Specific Test/Endpoint: CELL TRANSFORMATION
Test Category: MISCELLANEOUS CATEGORY
Specific Test/Endpoint: COMUTAGENESIS

Species / strain / cell type:

primary culture, other: Syrian hamster embryo cells; simian adenovirus SA7

Details on mammalian cell type (if applicable):

Test Object: MAMMAL,SYRIAN GOLDEN HAMSTER CELL CULTURE
Tissue Cultured: PRIMARY EMBRYO CELLS
Cells Observed: SOMATIC CELLS
Name of Agent (CAS RN): VIRUS,SIMIAN ADENOVIRUS SA7
METAL SALTS :ALUMINUM SULFATE ( 10043-01-3 )

Metabolic activation:

without

Test concentrations with justification for top dose:

0.6, 0.3, 0.15, 0.07 mM

Untreated negative controls:

yes

Remarks:

Syrian hamster embryo cells; simian adenovirus SA7

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

no

Details on test system and experimental conditions:

Taxonomic Name: MESOCRICETUS AURATUS
Test Object: MAMMAL,SYRIAN GOLDEN HAMSTER CELL CULTURE
Tissue Cultured: PRIMARY EMBRYO CELLS
Cells Observed: SOMATIC CELLS

Failure to enhance viral transformation after treatment of hamster cells with aluminium sulfate
Chemical Concentration (Mm) Surviving fraction SA7 foci Enhancement ratio
Al2 (SO4)2 0.6 0.74 18 1.2
0.3 0.83 16 1.0
0.15 1.02 16 0.8
0.07 1.04 15 0.8
0 1.00 19 1.0

Concentration. Chemical dilutions were added to mass cultures of HEC 18 hr before or 5 hr after SA7.Virus was absorbed for 3 hr, and the cells were transferred for survival (500 to 700 cells/dish) and for transformation assays (200,000 to 300,000 cells/dish).

Surviving fraction. Determined from plates receiving 500 to 700 cells. The number of colonies from virus- and chemical-treated cells was divided by the number of colonies from virus-inoculated control cells to give the surviving fraction. Cloning efficiency of control cells was from l0 to 15%.

SA7 foci. Number of foci from 106 plated cells.

Enhancement ratio Enhancement ratio was determined by dividing the TF of treated cells (TF = SA7 foci x reciprocal of the surviving fraction) by thatobtained from control cells. Numbers in italics are statistically significant at the 1% level.

Species / strain:

primary culture, other: Syrian hamster embryo cells; simian adenovirus SA7

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Transformation Assays.
Briefly, the procedure was as follows:
SA7 was added to HEC (3 to 4 x 107 plaque-forming units/culture) and adsorbed for 3 hr; the virus-inoculated cells were removed with trypsin (0.25% trypsin in MDM with 0.1 mM CaCl2), centrifuged, and resuspended to 106 cells/ml in MDM with 10% FBS and NaHCO3 (0.11 g/100 ml). The cells were vigorously mixed and plated into 60-mm dishes using 2 x 105 cells/dish; 3 ml of the above medium were then added to each plate. After incubation for 3 days, the medium was changed to MDM with 0.1 mM CaCl (4, 21), 10% FBS, and NaHCO3 (0.22 g/100 ml). After 6 days, transformation assay plates were overlaid with 3 ml of the above medium containing Bacto-agar (0.3 g/100 ml). At intervals of 4, 5, and 6 days, 3 ml of additional agar medium were added. Final focus counts were made 25 to 30 days from the beginning of the experiment.

Survival Assays. After resuspension of the HEC to 106 cells/mI (see “TransformationAssays”), the cells were diluted 1:300 in medium to give 333 cells/0.1 ml. Two-tenths ml (666 cells) was then added to each of 5 plastic dishes, followed by 3 ml of MDM with 10% FBS and NaHCO3 (0.11 g/100 ml). Five to 6 days later, 3 ml of medium with double the amount of NaHCO3were added to each plate, and after 8 to 9 days of total incubation the cell colonies were fixed in 10% buffered fonmalin and stained with 0.02% crystal violet. The cloning efficiency of virus-inoculated cells under these conditions was usually 8 to 12%.

Chemical Treatment. Fresh stock solutions of metal salts were prepared for each experiment by dissolving the salts in acetone: water (1:1). Appropriate dilutions were then made in the complete medium to give the desired final concentration. In each experiment, 2 plates of HEC were treated with metal salts for 18 hr prior to inoculation , on 5 to 10 plates were treated at 5 hr after virus inoculation. After pretreatment, HEC were rinsed with complete medium and inoculated with SA7. Transformation and clonal assays were then performed with each treatment group as de scnibed above. When treated after virus inoculation, the metal salts remained in the medium for 48 hr.

Determination of Enhancement.The fraction of cells surviving treatment was determined from assay plates which were seeded with 666 cells/plate. The number of colonies in 5 plates seeded with treated cells was divided by the number of colonies in 5 plates with control cells to give the surviving fraction of treated cells.
The total number of SA7 foci in 5 control plates, each receiving 200,000 cells, was used as the frequency of transformation per 106 virus-inoculated control cells. The frequency of transformation of treated cells (number of SA7 foci per 106 surviving cells) was calculated by multiplying the number of SA7 foci from 5 plates by the reciprocal of the surviving fraction of treated cells. Detailed methods for determining the frequency of transformation and enhance ment ratios using data from actual enhancement expeni ments have been presented (11, 12). Similar methods for calculation have been used for the determination of mutation frequency (14) and for the demonstration of enhancement of SV40 transformation by DNA base analogs (52) in mammalian cells.
Enhancement was expressed as the ratio between the TF of treated, surviving cells and the TF of control cells. Statistical significance was determined using a table of ratios (10) derived from the Lorenz table (29), which is based upon the Poisson distribution. The increased TF was considered statistically significant at the 5 or 1% confidence level if the enhancement ratio exceeded the appropriate value obtained from the Lonenz table.

Remarks on result:

other: strain/cell type: VIRUS,SIMIAN ADENOVIRUS SA7

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative Aluminium sulfate was negative for enhancement of viral transformation after treatment of hamster cells, when tested at up to 0.6 mM

Aluminium sulfate was negative for enhancement of viral transformation after treatment of hamster cells, when tested at up to 0.6 mM

Executive summary:

Aluminium sulfate was negative for enhancement of viral transformation after treatment of hamster cells, when tested at up to 0.6 mM

Chemical dilutions were added to mass cultures of HEC 18 hr before or 5 hr after SA7.Virus was absorbed for 3 hr, and the cells were transferred for survival (500 to 700cells/dish) and for transformation assays (200,000 to 300,000 cells/dish).

 

Surviving fraction was determined from plates receiving 500 to 700 cells. The numbe rof colonies from virus- and chemical-treated cells was divided bythe number of colonies from virus-inoculated control cells to givethe surviving fraction. Cloning efficiency of control cells was froml0to 15%.

Number of SA7 foci was from 10⁶plated cells.

Enhancement ratio was determined by dividing the TF oftreated cells (TF = SA7 foci x reciprocal of the surviving fraction)by that obtained from control cells. Numbers in italics are statistically significant at the 1% level.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Substance Aluminium sulphate was evaluated for its mutagenic and genotoxic potential in vitro and vivo.Overall the data summarised do not indicate any mutagenic or genotoxic potential of the substance.

In vitro Studies

Aluminium sulphate and both category sulfate were negative with and without metabolic activation in an Ames test performed according to OECD TG 471 (Wagner, 2001a/b); . Potassium sulfate and calcium sulfate were negative in an in vitro chromosomal aberration test with Chinese hamster ovary cells performed according to OECD TG 473 (Gudi, 2001a/b). Noin vitrogenotoxicity studies were available for potassium magnesium sulfate.

Aluminium sulphate was negative using Bacillus subtilis recombination assay (Kada et al. 1980, Kanematsu et al. 1980; Nishioka 1975

Aluminium sulphate was negative using "SOS Chromotest, a direct assay of induction of an SOS function in Escherichia coli K-12 to measure genotoxicity", Proc. Natl. Acad. Sci., 79, 5971 - 5975, Olivier, Ph. and Marzin, D. (1987)

ALTERNATIVE IN VITRO TESTS/ . In vitro experiments showed that the aluminum sulfate concentration needed to inhibit the enzyme activity was 1.0-5.0 mM (N = 3) in brain, 4.0-5.0 mM (N = 3) in liver and 0.0-5.0 mM (N = 3) in kidney. [Schetinger MR et al; Braz J Med Biol Res 32 (6): 761-766 (1999)]

In vivo Studies

An OECD TG 474 study was performed (micronucleusin vivo test, mouse) with the analogue substance calcium sulfate dihydrate, and reported that the substance tested negative in the micronucleus testin vivoup to the test concentration of 5000 mg/kg bw (SIAM 17, 2003).

ALTERNATIVE IN VIVO TESTS

In vivo treatment with aluminum sulfate did not inhibit ALA-D activity in the brain homogenate (Table 2). In liver, treatment with citrate and aluminum plus citrate increased ALA-D activity by 30-40% when compared to the control group. In kidney, ALA-D activity was inhibited 24% in the aluminum plus citrate group when compared to the control group. There was no difference between the control and citrate groups., Schetinger MR et al; Braz J Med Biol Res 32 (6): 761-766 (1999)

Justification for classification or non-classification

Based on the hazard assessmentof aluminium sulphate in section 2.1 and 2.2.in IUCLID 5.4., available data for the substance and following the “Guidance on Information Requirement and Chemical Safety Assessment R.8. Characterisation of dose [concentration]- response for human health” andaccording to the criteria described in Directive 67/548 and in the CLP Regulation:

 

 

Directive 67/548	Mutagenicity-Genetic Toxicity Muta. Cat. 1; R46 May cause heritable genetic damage. Muta. Cat. 2; R46 May cause heritable genetic damage. Muta. Cat. 3; R68 Possible risk of irreversible effects.
CLP	Germ cell mutagenicity Muta. 1A Muta. 1B Muta. 2 H340: May cause genetic defects <state route of exposure if it is conclusively proven that no other routes of exposure cause the hazard>. H341: Suspected of causing genetic defects <state route of exposure if it is conclusively proven that no other routes of exposure cause the hazard>.

 

It is concluded that the substance aluminium sulphate does not meet the criteria to be classified for human health hazards for Mutagenicity-Genetic Toxicity