Aluminium sodium tetrahydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Genotoxic effects of aluminum chloride in cultured human lymphocytes treated in different phases of cell cycle

Author:

Lima, P. D. L. et al.

Year:

2007

Bibliographic source:

Food and Chemical Toxicology 45: 1154-1159

Materials and methods

Principles of method if other than guideline:

The present study aimed to evaluate the mutagenic potential of aluminum chloride (AlCl3) in human lymphocyte cell cultures.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium chloride

EC Number:

231-208-1

EC Name:

Aluminium chloride

Cas Number:

7446-70-0

Molecular formula:

AlCl3

IUPAC Name:

aluminum trichloride

Details on test material:

- Name of test material (as cited in study report): Aluminum chloride AlCl3
- Analytical purity: no data

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

The culture medium consisted of 5 mL HAM-F10 (78%), heat-inactivated fetal calf serum (20%), phytohaemagglutinin-M (2%) and antibiotics 0.01 mg/mL of penicillin and 0.005 mg/mL of streptomycin. The culture tubes were incubated at 37 °C in a humidified atmosphere composed of 5% CO2 atmosphere and 95% humidity.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 5, 10, 15, 25 µM AlCl3

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: methanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Doxorubicin (0.01 µg/mL)

Details on test system and experimental conditions:

Cell culture processing & conditions:
Short-term lymphocyte cultures were initiated according to a standard protocol (reference provided: Preston et al., 1987) using 5mL HAM-F10 (78%, heat-inactivated fetal calf serum (20%), phytohemagglutinin-M (2%) and antibiotics 0.01mg/mL of penicillin, 0.005 mg/mL streptomycin. Cultures were maintained at 37ºC in a humidifed atmosphere composed of 5% CO2 and 95% humidity.

Preparation of test solutions:
Limited information. The article states that the test solutions were prepared by dissolution in methanol. The authors state (but do not provide the actual data to support the statement) that methanol did not reduce the mitotic index when compared to cultures without its presence and did not induce chromosomal aberrations.

Administration of test solutions:
Volume: NR; Method not reported.

Duration of exposure, schedule/duration of incubations:
For the G1 phase: lymphocytes were treated with a combination of 0.2mL PHA and AlCl3 and then incubated for 52 hours at 37ºC until fixation.

For the transition G1-S phase: cultures were treated with AlCl3 24 hours after stimulation with PHA and then incubated for 52 hours at 37ºC until fixation.

For the S phase:
Pulse treatments of AlCl3 were administered for 1 hour and 6 hours, 24 hours after PHA stimulation. After each pulse treatment, cells were washed once in serum-free medium, re-incubated in the complete medium for 52 hours prior to fixation.

For the G2 phase:
69 hour cultures were treated with AlCl3 for 3 hours and then fixed immediately, giving a total incubation time of 72 hours.

Analytical verification of dose levels:
Not carried out.

Incubations per dose/time point:
Unclear; appears to be single cultures but one from each donor? No measures of variability were provided.

Further details on study design:
Cells were fixed using colchicine (final concentration 0.0016%) at 50 hours and then harvested at 52 hours. The cells were harvested by centrifugation, treated with KCl at 37ºC for 20 minutes. The cells were then centrifuged again and fixed in 1:3 (v/v) acetic acid: methanol. The slides were then prepared, air-dried and stained with 3% Giemsa (ph=6.8) for 8 minutes.
No metabolic activation was applied.

Measurement of study outcomes:
Metaphases were examined using an optical microscope to enumerate the number of structural and mureical CAs. The frequency of CAs was determined in 100 metaphases per culture. The mitotic index was also determined (number of metaphases per 2000 lymphoblasts per culture).

Ancillary endpoints examined:
-cytotoxicity: The mitotic index was determined (number of metaphases per 2000 lymphoblasts per culture). Polyploidy and endoreduplication.

Evaluation criteria:

CA assay
The statistical significance of differences between treatment and control appears to be the criteria used to define a positive or negative response.

Gaps and breaks were included in the total chromosome aberration index. There is uncertainty concerning the actual types of structural aberrations included in the “total” index.

Statistics:

The student’s t-test was used to compare the frequencies of CAs observed in cells exposed to AlCl3 with the control.

One-way ANOVA (F-test)was used to test for significant differences in the mitotic index.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The MI in treated cultures was 32 to 61% of the negative control MI in the cells assessed in the G1 phase.

The MI was also significantly reduced relative to the negative control in all treated cultures in the G1/S phase. At 25 μM AlCl3, the MI was only 21% of the control.

The largest decreases in MI were observed in the S-phase cells exposed to pulse treatment of AlCl3 for 6 hours. The MI (expressed as a percentage of the negative control MI) were 25%, 18%, 9%, and 4% for the 5, 10, 15 and 25 μM AlCl3 concentrations, respectively.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome aberrations and mitotic index:

At G1 and G1/S, the frequency of CAs was significantly increased with all the tested concentrations of AlCl3. During G1 and G2 treatments, significantly induced endoreduplication and polyploidy were observed. AlCl3 treatment during S phase resulted in highly significant increases in the frequency of CAs in most concentrations (p<0.01); however, there were no significant differences between S phase treatments of 1 and 6 hour. The treatment at G2 also induced a significant increase in the frequency of CAs. Chromatid gaps and chromatid breaks were the most frequent CAs.

The cytotoxic effects of AlCl3 were observed as decreases in the MI of lymphocyte in cultures treated during the G1, G1/S, S and G2 phases of the cell cycle.

(*p<0.05, ** p<0.01)

G1 phase: chromosome aberrations per cell

Dose (μM)	MI (%)	Gaps	Breaks	Total	Poly	Endo
Contr	5.6	1	0	1	0	0
5	3.4*	4	6	10*	6	10*
10	2.4*	8	8	16*	10*	9*
15	1.9*	8	9	17*	9*	9*
25	1.8*	8	14	22*	14*	8*
DOX	3.1*	5	4	9*	2	4

 

G1/S phase: chromosome aberrations per cell

Dose (μM)	MI (%)	Gaps	Breaks	Total	Poly	Endo
Contr	5.5	2	0	2	0	1
5	2.8*	12	0	12*	0	0
10	1.9*	15	9	24*	1	0
15	1.5*	14	8	22*	0	1
25	1.2*	18	10	28*	1	0
DOX	3.2*	17	6	23*	5	0

 

S phase: chromosome aberrations per cell

(Pulse treatment for 1 hour)

Dose (μM)	MI (%)	Gaps	Breaks	Total	Poly	Endo
Contr	5.4	2	0	2	0	0
5	2.3*	28	8	36**	0	0
10	1.7*	35	11	46**	0	0
15	1.2*	30	14	44**	0	0
25	1.0*	43	19	62**	0	0
DOX	2.4*	10	7	17*	1	1

 

S phase: chromosome aberrations per cell

(Pulse treatment for 6 hours)

Dose (μM)	MI (%)	Gaps	Breaks	Total	Poly	Endo
Contr	4.9	2	0	2	0	0
5	1.4*	27	9	36**	0	0
10	1.0*	23	12	35**	0	0
15	0.5**	7	8	15*	0	0
25	0.2**	10	13	23*	0	0
DOX	1.9*	14	10	24*	3	0

 

G2 phase: chromosome aberrations per cell

Dose (μM)	MI (%)	Gaps	Breaks	Total	Poly	Endo
Contr	5.8	8	0	8	0	1
5	3.5*	15	4	19*	10*	2
10	3.0*	17	6	23*	14*	0
15	2.9*	23	8	31*	20*	4
25	2.4*	33	14	47**	27*	5
DOX	5.2*	24	8	32*	7	4

 

The authors state that “chromatid gaps and chromatid breaks” were the most frequent chromosome aberrations. However, it is unclear whether the “breaks” in the results tables presented in the article refer to chromatid breaks and/or chromosome breaks. The authors also included gaps in the total damage although gaps are not usually included in the total aberration frequency.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

The total aberrations (gaps plus breaks) were significantly higher in all treated cultures in cells in the G1 and G1/S phase, the S phase and also the G2 phase. During the G1 phase, the treated cultures also exhibited significantly increased polyploidy and endoreduplication compared with the negative control. The biological relevance of these results is unclear, however, due to the high cytotoxicity in the cultures.