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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on experimental data and read-across in a weight of evidence approach:

Bacterial gene mutation: negative

Gene mutation in mammalian cells: negative

Cytogenicity/chromosome aberration in mammalian cells: positive

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 Dec 2009 – 7 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (tk)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for spontaneous mutant frequency
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9) was obtained from Molecular Toxicology Incorporated, USA.
Test concentrations with justification for top dose:
Aluminium hydroxide:
6.094, 12.19, 24.38, 48.75, 97.5, 195, 390 and 780 µg/mL for experiments 1 and 2 in the presence and in the absence of metabolic activation.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.5% methylcellulose (MC)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9) and benzo(a)pyrene (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration:
+S9: 3 hours
-S9:
Aluminium hydroxide:
- Experiment 1 – 3 hours
- Experiment 2 – 3 hours
- Expression time (cells in growth medium): For an expression period of 2 days, the cultures were maintained in flasks, subculturing as necessary to try to maintain cell densities below 1 x 10E6 cells/mL and also to keep at least 1 x 10E7 cells per flask.

SELECTION AGENT (mutation assays): 5-trifluorothymidine ( final concentration of 3 µg/mL)

NUMBER OF REPLICATIONS: duplicates in 2 experiments

NUMBER OF CELLS EVALUATED: number of TFT-resistant mutants per 10E6 viable cells 2 days after treatment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Mutant frequency (MF): number of TFT-resistant mutants per 10E6 viable cells 2 days after treatment.

MF = - ln P(0)/(number of cells per well x (viability/100))

- where P(0) = (number of wells with no colony/total number of wells)

*The number of cells per well was 2000 on average on all mutant plates.

Criteria used to Assess Assay Validity:
1. The mean mutant frequencies in the negative (vehicle) control are in the normal range between 50 and 70 mutants per 106 viable cells.
2. At least one positive control should show either an absolute increase in mean total MF of at least 300E10-6 (at least 40% of this increase should be in small colony MF), or an increase in small colony mutant frequency of at least 150E10-6 above the vehicle control.
3. The RTG for the positive controls should be greater than 10%.
4. The mean cloning efficiencies of the negative controls between 65% and 120% on day 2.
5. The mean suspension growth of the negative controls between 8 and 32 following 3 hour treatments or between 32 and 180 following 24 hour treatments.
6. No excessive heterogeneity between replicate cultures.

Positive response definition:
A test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF [global evaluation factor].
2. The linear trend was positive.

“A test article was considered as positive in this assay if BOTH of the above criteria were met. A test article was considered as negative in this assay if neither of the above criteria were met. Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.”

The GEF for microwell assays is defined as 126 mutants per 10E6 viable cells.
Statistics:
The significance of increases in mutant frequencies in comparison with controls and the global evaluation factor [GEF] was assessed. The data were checked for a linear trend in mutant frequency with increasing treatment concentration using weighted regression. The test for linear trend was one-tailed.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and 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:
Aluminium hydroxide – Cytotoxicity Range-Finder Experiment
There were no appreciable differences in RTG and MF between the vehicle control and the untreated control.

Slightly greater toxicity (lower RTG value) was observed for controls subjected to the percoll differential centrifugation and cell recovery steps compared to controls not subjected to these steps.

These effects were considered minimal and acceptable, and there were no appreciable effects on the MF.

No significant changes in osmolality or pH were observed in the 3 and 24-hour range-finder experiment at the highest tested concentration (780 µg/mL) compared to the vehicle control.

Aluminium hydroxide: mutant frequencies (5-TFT resistant mutants/106viable cells, 2 days after treatment)

Dose (µg/ml)

Experiment 1

Experiment 2

 

-S9

+S9

-S9

+S9

0

64.78

59.07

53.44

43.62

UTC*

64.83

59.47

61.05

56.05

6.094

56.02

45.44

59.14

57.14

12.19

61.34

48.94

63.23

42.23

24.38

59.34

58.03

55.94

45.21

48.75

54.81

52.67

61.32

53.57

97.5

61.54

47.28

57.20

42.24

195

54.94

56.35

54.48

77.32

390

49.17

50.76

55.21

57.90

780

58.15

53.51

70.52

65.18

Linear trend

NS**

NS

NS

P<0.01

MMS/ B[a]P***

 

 

 

 

--/0.5

--

131.34

 

126.19

--/1

--

317.47

 

228.41

15/2

659.14

1047.7

441.10

525.12

20/3

755.10

878.38

593.79

883.47

*Untreated control

**Not significant

***MMS dose for -S9- or B[a]P dose for +S9

 

Mutant frequencies in both experiments at all tested concentrations were below the sum of the mean control mutant frequency plus the global evaluation factor (GEF). The observation of a significant linear trend in experiment 2 in the presence of S-9 without any corresponding increases in mutant frequencies approaching GEF was not considered a biologically relevant observation and the study was considered as providing a negative result for Al hydroxide.

 

The proportion of small colony mutants for the negative controls in the absence and presence of S-9 was 56% in experiment 1 and from 44% to 50% in experiment 2. Increased colony mutants were observed for the positive control substances.

 

Aluminium chloride

Experiment 1 (3-hour treatment). Mutant frequencies (5-TFT resistant mutants/106viable cells 2 days after treatment)

Dose

(µg/ml)

-S9

+S9

0

91.54

70.46

3.125

70.68

64.03

6.25

56.47

59.90

12.5

68.44

58.97

25

74.50

56.35

50

82.67

69.97

Linear trend

NS*

NS

MMS/ B[a]P**

 

 

15/2

699.96

934.97

20/3

1095.71

1650.41

*Not significant

**MMS dose for -S9 or B[a]P dose for +S9

 

Experiment 2 (24-hour treatment in the absence and 3-hour treatment in the presence of S-9). Mutant frequencies (5-TFT resistant mutants/106viable cells 2 days after treatment)

Dose

(µg/ml)

-S9

Dose

(µg/ml)

+S9

0

52.63*

0

99.69

5

48.60

5

78.60

10

41.47

10

94.77

20

42.54

15

91.54

40

48.29

20

110.31

60

38.88

25

75.33

80

33.66

30

113.59

100

36.18

40

81.95

120

52.27

50

97.03

Linear trend

NS**

 

NS

MMS/ B[a]P***

 

 

 

5/2

962.16

 

1077.54

7.5/3

1246.55

 

887.11

*Based on one replicate only

**Not significant

***MMS dose for -S9 or B[a]P dose for +S9

 

In experiments 1 and 2 mutation frequencies at all tested concentrations were less than the sum of the mean control mutation frequency plus the GEF with a negative linear trend, indicating a negative result.

 

The proportion of small colony mutants for the negative controls in the absence and presence of S-9 were from 44% to 60% in experiment 1 and from 51% to 58% in experiment 2. Marked increases in the number of small and large colony mutants were observed after treatment with positive control chemicals.

Conclusions:
Al hydroxide did not induce mutation at the tk locus of L5178Y mouse lymphoma cells under the conditions of these experiments.
Executive summary:

Covance (2010b) reported negative findings from their assay of forward mutations at the tk locus of L5178Y mouse lymphoma cells. They conducted two experiments with Al(OH)3, each with a 3 hour treatment duration. Two experiments were also conducted with AlCl3, one with a 3-hour and the other with a 24-hour treatment duration. Each experiment included incubations with and without metabolic activation. Concentrations of the test items were selected based on results of range-finding studies and observation of precipitation in the incubations. In the Al(OH)3 experiments, eight concentrations ranging from 6.094 µg/mL to 780 µg/mL were used for determination of mutation frequencies. For AlCl3, results from five concentrations (from 3.125 to 50 µg/mL) were used in the experiments with 3-hour treatment duration both in the presence and absence of metabolic activation. In the experiments with 24 hour duration treatments, 8 concentrations were used: from 5 to 120 µg/mL in the absence of metabolic activation and from 5 to 50 µg/mL in the presence of metabolic activation.Negative (vehicle) and positive (methyl methane sulphonate with S9 activation; benzo-[a]-pyrene without S9 activation) controls were included in each experiment.The results of the experiments with AlCl3 were negative: the mutant frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF).A negative linear trend was also observed. In the experiments with Al(OH)3, the mutation frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus GEF. Although a significant positive linear trend in mutation frequencies was observed in the presence of S-9 in one of the experiments with Al(OH)3, no corresponding increase in mutant frequencies approaching the GEF was observed, and the effect was not observed in the other experiment; therefore, this observation was not considered biologically relevant. This study was conducted in accordance with negative. This study is well described, was conducted in accordance with OECD TG #476 (1997), and with other guidelines, and complied with principles of Good Laboratory Practice.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 Dec 2009 – 7 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (tk)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for spontaneous mutant frequency
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9) was obtained from Molecular Toxicology Incorporated, USA.
Test concentrations with justification for top dose:
Aluminium chloride:
Experiment 1, Concentrations assayed: 3.125, 6.25, 12.5, 25 and 50 µg/mL in the presence and in the absence of metabolic activation.
Experiment 2, Concentrations assayed: 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL in the absence of metabolic activation
5, 10, 15, 20, 25, 30, 40 and 50 µg/mL in the presence of metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water for aluminium chloride
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9) and benzo(a)pyrene (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration:
+S9: 3 hours
-S9:
Aluminium chloride
- Experiment 1 – 3 hours
- Experiment 2 – 24 hour
- Expression time (cells in growth medium): For an expression period of 2 days, the cultures were maintained in flasks, subculturing as necessary to try to maintain cell densities below 1 x 10E6 cells/mL and also to keep at least 1 x 10E7 cells per flask.

SELECTION AGENT (mutation assays): 5-trifluorothymidine ( final concentration of 3 µg/mL)

NUMBER OF REPLICATIONS: duplicates in 2 experiments

NUMBER OF CELLS EVALUATED: number of TFT-resistant mutants per 10E6 viable cells 2 days after treatment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Mutant frequency (MF): number of TFT-resistant mutants per 10E6 viable cells 2 days after treatment.

MF = - ln P(0)/(number of cells per well x (viability/100))

- where P(0) = (number of wells with no colony/total number of wells)

*The number of cells per well was 2000 on average on all mutant plates.

Criteria used to Assess Assay Validity:
1. The mean mutant frequencies in the negative (vehicle) control are in the normal range between 50 and 70 mutants per 106 viable cells.
2. At least one positive control should show either an absolute increase in mean total MF of at least 300E10-6 (at least 40% of this increase should be in small colony MF), or an increase in small colony mutant frequency of at least 150E10-6 above the vehicle control.
3. The RTG for the positive controls should be greater than 10%.
4. The mean cloning efficiencies of the negative controls between 65% and 120% on day 2.
5. The mean suspension growth of the negative controls between 8 and 32 following 3 hour treatments or between 32 and 180 following 24 hour treatments.
6. No excessive heterogeneity between replicate cultures.

Positive response definition:
A test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF [global evaluation factor].
2. The linear trend was positive.

“A test article was considered as positive in this assay if BOTH of the above criteria were met. A test article was considered as negative in this assay if neither of the above criteria were met. Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.”

The GEF for microwell assays is defined as 126 mutants per 10E6 viable cells.
Statistics:
The significance of increases in mutant frequencies in comparison with controls and the global evaluation factor [GEF] was assessed. The data were checked for a linear trend in mutant frequency with increasing treatment concentration using weighted regression. The test for linear trend was one-tailed.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and 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:
RANGE-FINDING/SCREENING STUDIES:
Aluminium chloride – Cytotoxicity Range-Finder Experiment:
No significant changes in osmolality or pH were observed in the 3 and 24-hour range-finder experiment at the highest tested/analysed concentration (83.38 µg/ml) compared to the vehicle control.

Aluminium hydroxide: mutant frequencies (5-TFT resistant mutants/106viable cells, 2 days after treatment)

Dose (µg/ml)

Experiment 1

Experiment 2

 

-S9

+S9

-S9

+S9

0

64.78

59.07

53.44

43.62

UTC*

64.83

59.47

61.05

56.05

6.094

56.02

45.44

59.14

57.14

12.19

61.34

48.94

63.23

42.23

24.38

59.34

58.03

55.94

45.21

48.75

54.81

52.67

61.32

53.57

97.5

61.54

47.28

57.20

42.24

195

54.94

56.35

54.48

77.32

390

49.17

50.76

55.21

57.90

780

58.15

53.51

70.52

65.18

Linear trend

NS**

NS

NS

P<0.01

MMS/ B[a]P***

 

 

 

 

--/0.5

--

131.34

 

126.19

--/1

--

317.47

 

228.41

15/2

659.14

1047.7

441.10

525.12

20/3

755.10

878.38

593.79

883.47

*Untreated control

**Not significant

***MMS dose for -S9- or B[a]P dose for +S9

 

Mutant frequencies in both experiments at all tested concentrations were below the sum of the mean control mutant frequency plus the global evaluation factor (GEF). The observation of a significant linear trend in experiment 2 in the presence of S-9 without any corresponding increases in mutant frequencies approaching GEF was not considered a biologically relevant observation and the study was considered as providing a negative result for Al hydroxide.

 

The proportion of small colony mutants for the negative controls in the absence and presence of S-9 was 56% in experiment 1 and from 44% to 50% in experiment 2. Increased colony mutants were observed for the positive control substances.

 

Aluminium chloride

Experiment 1 (3-hour treatment). Mutant frequencies (5-TFT resistant mutants/106viable cells 2 days after treatment)

Dose

(µg/ml)

-S9

+S9

0

91.54

70.46

3.125

70.68

64.03

6.25

56.47

59.90

12.5

68.44

58.97

25

74.50

56.35

50

82.67

69.97

Linear trend

NS*

NS

MMS/ B[a]P**

 

 

15/2

699.96

934.97

20/3

1095.71

1650.41

*Not significant

**MMS dose for -S9 or B[a]P dose for +S9

 

Experiment 2 (24-hour treatment in the absence and 3-hour treatment in the presence of S-9). Mutant frequencies (5-TFT resistant mutants/106viable cells 2 days after treatment)

Dose

(µg/ml)

-S9

Dose

(µg/ml)

+S9

0

52.63*

0

99.69

5

48.60

5

78.60

10

41.47

10

94.77

20

42.54

15

91.54

40

48.29

20

110.31

60

38.88

25

75.33

80

33.66

30

113.59

100

36.18

40

81.95

120

52.27

50

97.03

Linear trend

NS**

 

NS

MMS/ B[a]P***

 

 

 

5/2

962.16

 

1077.54

7.5/3

1246.55

 

887.11

*Based on one replicate only

**Not significant

***MMS dose for -S9 or B[a]P dose for +S9

 

In experiments 1 and 2 mutation frequencies at all tested concentrations were less than the sum of the mean control mutation frequency plus the GEF with a negative linear trend, indicating a negative result.

 

The proportion of small colony mutants for the negative controls in the absence and presence of S-9 were from 44% to 60% in experiment 1 and from 51% to 58% in experiment 2. Marked increases in the number of small and large colony mutants were observed after treatment with positive control chemicals.

Conclusions:
Al chloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells under the conditions of these experiments.
Executive summary:

Covance (2010b) reported negative findings from their assay of forward mutations at the tk locus of L5178Y mouse lymphoma cells. They conducted two experiments with Al(OH)3, each with a 3 hour treatment duration. Two experiments were also conducted with AlCl3, one with a 3-hour and the other with a 24-hour treatment duration. Each experiment included incubations with and without metabolic activation. Concentrations of the test items were selected based on results of range-finding studies and observation of precipitation in the incubations. In the Al(OH)3 experiments, eight concentrations ranging from 6.094 µg/mL to 780 µg/mL were used for determination of mutation frequencies. For AlCl3, results from five concentrations (from 3.125 to 50 µg/mL) were used in the experiments with 3-hour treatment duration both in the presence and absence of metabolic activation. In the experiments with 24 hour duration treatments, 8 concentrations were used: from 5 to 120 µg/mL in the absence of metabolic activation and from 5 to 50 µg/mL in the presence of metabolic activation.Negative (vehicle) and positive (methyl methane sulphonate with S9 activation; benzo-[a]-pyrene without S9 activation) controls were included in each experiment.The results of the experiments with AlCl3 were negative: the mutant frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF).A negative linear trend was also observed. In the experiments with Al(OH)3, the mutation frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus GEF. Although a significant positive linear trend in mutation frequencies was observed in the presence of S-9 in one of the experiments with Al(OH)3, no corresponding increase in mutant frequencies approaching the GEF was observed, and the effect was not observed in the other experiment; therefore, this observation was not considered biologically relevant. This study was conducted in accordance with negative. This study is well described, was conducted in accordance with OECD TG #476 (1997), and with other guidelines, and complied with principles of Good Laboratory Practice.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
The human lymphocytes were obtained from heparinized whole blood samples provided by two, young (actual age not specified), healthy (not further specified), non-smoking donors.

- Type and identity of media: RPMI 1640 containing 20% fetal calf serum
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 500, 1000, 2000 and 4000 μM Al2(SO4)3 (equivalent to 0, 1000, 2000, 4000, and 8000 μM Al).
Vehicle / solvent:
Water
Untreated negative controls:
no
Remarks:
Only one harvest time was used for which a vehicle control culture was conducted.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: 0.51 µM
Untreated negative controls:
no
Remarks:
Only one harvest time was used for which a vehicle control culture was conducted.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Griseofulvin (43 µM)
Details on test system and experimental conditions:
Cell culture processing & conditions:
Whole blood samples were stimulated with phytohemoagglutinin (PHA 1.5%) and cultured in RPMI 1640 containing 20% fetal calf serum and antibiotics (5 UI/ml penicillin and 5μg/mL streptomycin). Each culture was conducted in duplicate. Cultures were maintained at 37 ºC.

Preparation of test solutions:
Limited information. The article states that the test compounds were dissolved in water, except CH3HgCl and griseofulvin which were dissolved in DMSO (1% of the final volume).

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

Duration of exposure, schedule/duration of incubations:
Test solutions were administered to the cultures 24 hours after PHA stimulation.
Incubation Duration: Incubations lasted until 72 hours when the cells were harvested.

Analytical verification of dose levels:
Not carried out.

Incubations per dose/time point: 2

Further details on study design:
MN were analyzed using a fluoresecent centromeric probe purchased from Oncor Inc. (Gaithersburg, MD. The author referred to Migliore et al. (1996) for details of the method (Migliore et al., 1996 – Mutagenesis 1996; 11(3): 285-290 – “The alphoid centromere-specific DNA probe used was ONCP 5095 (Oncor, USA). Slides were pretreated with a 10% pepsin solution (Sigma) in 10 mM HCL for 10 min at 37°C. After two washes in PBS and PBS/50 mM MgCl2, slides were postfixed with 1% formaldehyde in PBS/50 mM MgCl2 for 10 min at room temperature, rinsed in PBS, dehydrated via a -20°C ethanol series and air-dried. Denaturation of slides was performed in 70% formamide, 2X SSC (saline-sodium citrate buffer), pH 7.0, at 70°C for 2 min, followed by dehydration. After heating at 70°C for 5 min, 33 µL of probe were placed on the denatured slide under a coverslip and incubated overnight at 37°C in a moist chamber. Post-hybridization washes were performed, first in a solution of 50% formamide, 2X SSC, pH 7.0, at 37°C for 20 min, twice in 2X SSC for 4 min each, and then in 4X SSC, 0.05% Tween 20 (SSCW) for 5 min, both at 37°C. To minimize the background, the slides were preincubated for 10 min at 37°C in 4X SSC, 5% non-fat dry milk as immunological buffer (IB). For detection of the biotin labeled probe, fluorescein isothiocyanate (FTTC)-avidin conjugate (Pierce, Rockford, USA) and biotinylated goal anti-avidin (Pierce) were diluted in IB and alternately incubated for 30 min at 37°C at final concentrations of 5 and 0.5 µg/ml respectively. Three incubation steps were performed, each followed by three 2 min washes in SSCW at 37°C. After dehydrating through an ethanol series, slides were counterstained with 0.5 µg/ml propidium iodide (Sigma) dissolved in glycerol/DABCO (Sigma) as antifade solution. To evaluate probe hybridization efficiency (fluorescence site located at the centromere of chromosomes), metaphase spreads were also examined. Hybridization conditions were the same as previously described, except for slide pretreatment, which was performed with a solution of 200 µg/ml RNase (Sigma), 2X SSC at 37°C for 1 h, followed by two washes in 2X SSC.”

Measurement of study outcomes:
Coded slides were scored for MN analysis using at least 2000 binucleated cells per data point and individual. The results were expressed as the average number of micronucleated cells ± SD from two observations of 1000 cells on two different slides from two duplicate culture tubes.

Draft TG 487: cytoB-treated cultures – “micronucleus frequencies should be analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration or at least 2000 binucleated cells per concentration for a single culture.)” “Cells containing more than two main nuclei should not be analysed for micronuclei as the baseline frequency may be higher in these cells.”

Ancillary endpoints examined:
- cytotoxicity
Not assessed.

- cell proliferation (required to ensure treated cells have undergone mitosis during the assay)
- assessed using the % binucleated cells, a mesure of proliferation, counting 1000 lymphocytes for each experimental point.


Centromere positive and centromere negative micronuclei using an FTC-labelled MN as a centromere-positive MN (MNC+)and a non-labelled MN (MNC-)as a centromere-negative MN.
Evaluation criteria:
MN frequency:
Results were presented for the donors individually. Statistical significance using Fisher’s exact test was interpreted.

MNC+, MNC-:
Relative frequencies were determined and compared between treated and control cultures using Fisher’s exact test.
Statistics:
Fisher’s exact test was used to compare the frequency of micronucleated binucleated cells between the treated and control groups.
Fisher’s exact test was also used to compare the proportions of centromere positive and negative MN between the treated and control groups.
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Percentage of binucleated cells: See table below. The results do not indicate that toxicity may contribute to the positive results.

The authors consider the responses of aluminium sulphate, cadmium chloride, methyl mercuric chloride and potassium antimonate to be clearly positive. A difference in susceptibility of the two donors to micronuclei formation was evident.

The statistical significance of any dose-response was not tested.

Donor A(2000 BN scored per dose)

Dose (μM) (Al2(SO4)3

MN BN

MN BN/

1000,

mean(sd)

BN (%)

Contr

12

6.0 (1.4)

33.0

500

14

7.0 (2.8)

21.2

1000

23

11.5 (2.1)*

25.0

2000

30

15.0 (1.4)**

27.5

4000

11

5.5 (0.7)

29.5

* p<0.05, ** p<0.01, *** p<0.001 for comparison with control

 

Donor B(2000 BN scored per dose)

Dose (μM) (Al2(SO4)3

MN BN

MN BN/

1000,

mean(sd)

BN (%)

Contr

8

4.0 (1.4)

45.6

500

19

9.5 (3.5)*

47.2

1000

28

14.0 (0.0)***

46.7

2000

20

9.9 (3.0)*

53.2

4000

18

9.0 (0.0)*

33.9

* p<0.05, ** p<0.01, *** p<0.001 for comparison with the control.

 

Relative proportions of centromere positive and centromere negative micronuclei were presented in the article as a graph only. Aluminium induced relatively higher percentages of MNC+. The results do not however provide clear evidence to differentiate between an aneugenic or clastogenic effect.

 

The MN frequencies in the negative control group did not raise concern (<10/1000).

Conclusions:
positive without metabolic activation

Aluminium sulphate at doses of 1000, 2000, 4000 and 8000 μM of aluminium led to the induction of micronuclei in in-vitro cultures of human peripheral lymphocytes. Increases were more than 2-fold at concentrations of 2000 and 4000 μM Al in cells from both of the donors.
Executive summary:

Migliore et al. (1999) observed increases in MN at Al2(SO4)3 concentrations greater than 2000 µM-Al. Cytochalasin B (cytoB) was used as a cytokinesis block in the assay. Mitomycin C, a clastogen, and griseofulvin, an aneuploidogen, were used as positive controls and sufficient binucleated cells were scored to satisfy OECD Draft Guideline #487.

Blood samples were obtained from two young, healthy donors. Concentrations tested were 0, 100, 2000, 4000, 8000 µM-Al. Test solutions were administered to the cultures 24 hours after PHA stimulation followed by a 48 hour incubation period. Metabolic activation was not used. The % of binucleated cells was evaluated as a measure of proliferation. No obvious toxicity was observed. There was evidence of an increase in MN frequency with doses up to 2000 µM-Al followed by a decrease. At 2000 and 4000 µM, a 2-fold increase in MN frequencies was observed in both donors. Levels of MN in the control incubations were less than 1%. Production of both centromere negative and centromere positive MN was observed consistent with both clastogenic and aneuploidogenic potential, respectively. 

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
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: Tice, R.R. et al., 2000 and Burlinson, B. et al., 2007
Qualifier:
no guideline available
Principles of method if other than guideline:
The Comet Assay depends on the abaility of negatively-charged fragments of DNA to undergo electrophoresis in an agarose gel. The extent to which DNA migrates correlates directly with the amount of DNA damage. A suspension of cells is mixed with low melting point agarose, spread on a slide and lysed. DNA is then unwound and undergoes electrophoresis at a particular pH. The pH used dictates the types of DNA damage detected. For example, a neutral pH (7-8), as used in this study, detects predominantly double strand breaks and cross links while a pH>13 allows detection of single strand breaks, incomplete excision repair sites, and alkali labile sites (ALS) in addition to the lesions detected at neutral pH. Under the influence of an electric field, the damaged DNA migrates towards the anode producing a shape like a comet. The amount of migration and shape of the comet are an index of the DNA damage that can then be analysed visually or with image analysis software.
GLP compliance:
not specified
Type of assay:
comet assay
Target gene:
Not applicable.
Species / strain / cell type:
other: CD4+T cells
Details on mammalian cell type (if applicable):
Species/Cell Line/Source of Cells:
Human CD4+ T cells were obtained from a T-helper lymphoma Jurkat cell line from American Type Culture Collection (Manassas, VA, USA).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 50, 100, 500, 1000 and 5000 μM AlCl3.
Vehicle / solvent:
No information.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
Cell culture processing & conditions:
Cells were cultures in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) at 37 ºC in a atmosphere composed of 0.5% CO2. After they became confluent (every 3 to 4 days), the cultures were subcultured. Cells were washed twice with PBS and resuspended in 24 well-plates containing fresh medium and the test solutions.

Preparation of test solutions:
No information.

Administration of test solutions:
Volume: NR; Exposure occurred in well-plates.

Duration of exposure, schedule/duration of incubations:
The cells were challenged by exposure to metal solutions for 48 hours.

Analytical verification of dose levels:
Not carried out.

Incubations per dose/time point:
This was not stated for the Comet Assay. Apoptosis and viability tests were carried out in trplicate and prolferation measurement in quadruplicate.

Measurement of study outcomes:
Neutral Comet Assay
The comet assay (Trevigen, Gaithersburg, MD) was used. The authors report following manufacturer protocols. The metal-challenged Jurkat T-lymphocytes were cast in molten agarose (37 ºC) on glass slides (1 x 10E5 cells/mL at 1:10 ratio), lysed and unwound using alkaline (pH > 13) solutions. The gels then underwent electrophoresis (10 min at 22 mV). A stain (SYBR Green I nucleic acid gel stain) was added to show the fluorescent tails of DNA fragments that had migrated. The amount of DNA damage was measured by the comet tail length and normalized to untreated controls to give a DNA damage index (IDD).

50 different cells from two separate slides at each concentration were examined for each metal and the images analysed using a publicly available image analysis program for the Comet Assay.


Evaluation criteria:
Neutral Comet Assay
The length of the comet tail was used as an index of DNA damage and normalized to the value in the untreated control. An IDD (index of DNA damage) >75 was considered a high (“significant”) level of DNA damage. An IDD <75 but >0 was moderate damage and an IDD=0 was defined as low DNA damage.

Apoptosis (flow cytometry/caspase-9)
“Significant” apoptosis: ≥ 50% caspase-9 positive cells.

Significant detriment to viability or ‘toxicity”:
>50% propidium iodide positive cells.

Proliferation inhibition [3H-thymidine uptake]:
P<0.05 comparing metal-treated cells counts per minute with the untreated control.
Statistics:
Not described.
-Legend of figure 1 indicates the use of Student’s t-test and a significance level of p<0.05.
Species / strain:
other: CD4+T cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
- cytotoxicity
Cell viability was determined using propidium iodide (PI) staining and in a FACScan flow cytometer (Becton Dickinson Co.) General viability/toxicity for the different metal treatments were ranked using an LC50 index (half lethal concentration, i.e., the concentration at which 50% of the cells were viable). These tests were conducted in triplicate.

- apoptosis
For each metal, approximately 1 x 10E6 cells were incubated for 48 hours in a 24-well culture plate in 1 mL of media. Ten microlitres of a FITC-conjugated pan-caspase inhibitor (ApoStat) was added during the last 30 min. Cells were stained, harvested, washed twice with PBS, and resuspended in 400 µL of buffer for flow cytometry to quantify intracellular caspase-9 activity (FACScan flow cytometer - Becton Dickinson Co., San Diego, CA), Scatter gates were set to exclude cellular debris. All tests were conducted in triplicate.

- proliferation assays
- [3H]-thymidine incorporation
Quadruplicate proliferation assays were performed for 6 days using 96-well culture plates (Sigma), with a density of about 0.2 x 10E6 cells/ well for 6 days in 150 µL/well of complete media (DMEM, 10% FBS). The temperature was 37 ºC and the atmosphere had 0.5% CO2.. [3H]-Thymidine (1 mCi/well) was added during the last 12 h of the 6-day culture period and a cell harvester was used to collect the CD4+ T lymphocyte Jurkat cell membranes. A Beta plate counter was used to measure the [3H]-thymidine incorporation.

Primary Endpoint(s):

Neutral Comet Assay

Significant genotoxic effects were not observed at any of the concentrations tested (50 to 5000 µM-Al).

Apoptosis (flow cytometry/caspase-9)

Significant apoptosis was observed only at the highest concentration tested (5000 µM-Al)

 

Significant detriment to viability or ‘toxicity”:

Significant toxicity was not observed at any of the concentrations tested (50 to 5000 µM-Al).

 

Proliferation inhibition [3H-thymidine uptake]:

Significant inhibition of proliferation was not observed at any of the concentrations tested (50 to 5000 µM-Al).

Other salts:

Metal Chloride Concentrations (µM) at which Significant Harmful Effects (as defined in the article) were observed:

Metal

DNA damage

Apoptosis (Caspase-9)

Viability (PI)

Prolife-ration Inhibition

V

50

50

1000

50

Ni

50

100

5000

500

Co

5000

5000

500

100

Cu

>5000

500

5000

100

Nb

>5000

500

500

>5000

Mo

>5000

1000

>5000

500

Zr

5000

500

5000

>5000

Be

>5000

5000

1000

5000

Cr

>5000

>5000

>5000

>5000

Al

>5000

5000

>5000

>5000

Fe

>5000

5000

>5000

>5000

Conclusions:
Aluminium did not result in inhibition of cell growth (3H-thymidine uptake), significant increases in DNA double strand breaks (neutral Comet Assay), or viability (PI dye that stains nuclear DNA) at any concentration applied. A significant apoptotic effect determined through measurement of intracellular caspase-9 activity was observed only at the highest concentration of AlCl3 used (5000 µM-Al). Significant genotoxic effects were observed for vanadium, nickel and cobalt. Based on DNA damage, apoptosis, and effects on viability and prolfieration, the authors ranked the metal chloride salts as follows for harmful effects: V>Ni>Co>Cu>Nb>Mo>Zr>Be>Cr>Al>Fe.
Executive summary:

Caicedo et al. (2008) examined DNA damage in human (Jurkat) T-cells at a range of concentrations of AlCl3 (50, 100, 500, 1000 and 5000 µM-Al) using the neutral Comet Assay. Unwinding and electrophoresis of DNA at neutral pH detects fewer types of DNA damage compared with use of pH > 13. The neutral assay detects predominantly double strand breaks. Cell viability was measured using propidium iodide staining, cell proliferation using 3H-thymidine uptake and apoptosis using caspase-9 immunostaining and flow cytometry. A significant effect for DNA damage was defined as an index of DNA damage (based on relative comet tail length) of >75. More than 50% caspase-positive cells was considered significant apoptosis and >50% propidium iodide positive cells as a significant effect on viability. If the p-value from the Student’s t-test was less than 0.05 in the comparison of the counts per minute in metal-treated cells compared to untreated controls, the inhibition of proliferation based on 3H-thymidine uptake was considered significant. Aluminium did not result in significant inhibition of proliferation, significant increases in DNA double strand breaks, or a significant effect on viability at any concentration applied.

Apoptosis was significant only at 5000 µM-Al. The cells used in this study have not been extensively used with this assay. The responses observed are thus of unclear biological significance. The pH used for lysing and unwinding was also not clear from the report in the article. Overall the study appeared to follow GLP and the results were negative but of only limited relevance.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
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.
Qualifier:
no guideline available
Principles of method if other than guideline:
The Comet Assay depends on the ability of negatively-charged fragments of DNA to undergo electrophoresis in an agarose gel. The extent to which DNA migrates correlates directly with the amount of DNA damage. A suspension of cells is mixed with low melting point agarose, spread on a slide and lysed. DNA is then unwound and undergoes electrophoresis at a particular pH. The pH used dictates the types of DNA damage detected. For example, a neutral pH (7-8) detects predominantly double strand breaks and cross links and a pH>13, as used in this study, is more sensitive and allows detection of single strand breaks, double strand breaks, incomplete excision repair sites and also alkali labile sites (ALS). Under the influence of an electric field, the damaged DNA migrates towards the anode producing a shape like a comet. The amount of migration and shape of the comet are an index of the DNA damage that can be analysed visually or with image analysis software.
GLP compliance:
not specified
Type of assay:
comet assay
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Species/Cell Line/Source of Cells:
The human lymphocytes were obtained from whole blood samples provided by “healthy donors” aged between 19 and 30. The cells were isolated by Histopaque-1077 density gradient centrifugation.

Medium:
0.5 mL of lymphocyte suspension was added to 4.5 mL RPMI 1640 medium supplemented with fetal calf serum (20%), 2 mM L-glutamine, phytohemagglutinin-M (10 μg/mL), and antibiotics (100 U/mL of penicillin, 100 μg/mL streptomycin).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0 (vehicle control: 0.9% saline), 1, 2, 5, 10 or 25 μg AlCl3/mL, corresponding to 0, 4.15, 8.3, 20.75, 41.5 and 103.75 μM AlCl3.
Vehicle / solvent:
0.9% saline
Untreated negative controls:
other: It is not clear whether the negative control was a vehicle control or just for conditions.
Negative solvent / vehicle controls:
other: It is not clear whether the negative control was a vehicle control or just for conditions.
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
Cell culture processing & conditions:
The medium was 4.5 mL RPMI 1640 medium supplemented with fetal calf serum (20%), 2 mM L-glutamine, phytohemagglutinin-M (10 μg/mL), and antibiotics (100 U/mL of penicillin, 100 μg/mL streptomycin).Cultures were maintained at 37 ºC in an atmosphere composed with 5% CO2 .

Preparation of test solutions:
Limited information – The test substance was dissolved in 0.9% saline.

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

Duration of exposure, schedule/duration of incubations:
Comet Assay
The peripheral blood lymphocytes isolated by the Histopaque-1077 density gradient were exposed to AlCl3 for the complete 72 hour duration of the incubation.

Analytical verification of dose levels:
Not carried out.

Incubations per dose/time point:
Single cultures from three donors were carried out independently. Therefore, there were three cultures per dose/time point but no duplicates for an individual donor.

Measurement of study outcomes:
Alkaline Comet Assay
The authors cite Singh et al. (1991) for their methods. Slide preparation was conducted in the dark.

100 μL of the cell suspension was mixed with 200 μL of 2% low melting temperature agarose at 37 ºC, placed on a slide pre-coated with 0.5% normal melting temperature agarose, covered with a coverslip, and kept at 4 ºC for 5 minutes for the solidification of the agarose. Cells were lysed for 1 hour using 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100 (pH=10). After washing with re-distilled water, the slides were placed in the electrophoresis chamber, the chamber filled with the cold alkaline buffer (pH=13; 1 mM EDTA, 300 mM NaOH) and slides left for 40 minutes at 4 ºC. Electrophoresis was then performed for 25 minutes (1 V/cm, 300 mA). After washing three times with 0.4 M Tris buffer (pH=7.5), the slides were stained with 1 μM DAPI for 24 hours before examination using a fluorescence microscope (NIKON Eclipse 400) with a video camera. The CASP software was used to analyze the comets.

At least 50 cells per donor/dose were scored and the Olive Tail Moment calculated.

Oxidative Damage: Modified Comet Assay
Endo III (endonuclease III) is a purified oxidative DNA damage enzyme that catalyses the excision of oxidised pyrimidines. Fpg (formamidopyrimidine DNA glycolsylase) catalyses the excision of oxidized purines. The isolation, exposure and incubation steps used were the same as for the alkaline Comet Assay. Slide preparation followed the same steps outlined above except for washing the slodes three times for 5 minutes with endonuclease buffer (40 mL HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL bovine serum albumin, pH=8.0) after lysing. 50 μL aliquots of Endo III of Fpg were then added, the slide covered with a coverslip and incubated at 37 ºC for 45 minutes. The slides were then washed, stained and imaged as described above.

Ancillary endpoints examined:
Cytotoxicity

Apoptosis
The level of DNA damage and the freqeuncy of apoptosis were determined on the same slides using the Comet Assay. The apoptotic index was calculated as the percent of cells with diffuse fan-like tails and small heads from a minimum of 100 cells per donor/dose.

Apoptosis was also determined by flow cytometry using the annexin V-FITC apoptosis detection Kit I (BD Pharmingen, USA) and propidium iodide in lymphocytes from a SINGLE donor. Samples were analyzed using a FACscan flow cytometer (Becton Dickinson) and a computer system for data acquisition and storage. Three different cell populations were discriminated on the basis of forward and sideways light scattering: early apoptotic cells than fluoresced green (annexin+/IP-), necrotic cells that fluoreseced orange (annexin-/IP-+) and late apoptotic cells that were positive for both annexin V-FITC and PI (annexin+/IP+).

Other endpoints:
Cell-cycle distribution of effects
Analysis of the cell-cycle distribution of effects detected by the Comet Assay was conducted using the cells from two donors. The authors state that the computerized imaging system of the CASP software allows the measurement of the total fluorescence intensity (i.e. the relative DNA content) of individual cells permitting the distinction of cells in different phases of the cell cycle. Fifty randomly selected cells from each experiment/dose group were evaluated for total fluoresecence (i.e. relative DNA content as an index of the stage of the cell cycle) and DNA damage.

Kinetics of DNA repair
A culture treated with 10μg/mL of AlCl3.6H2O for 72 hours as described for the other experiments was then irradiated on ice with 2 Gy using a Co-60 source (Siemens Theratron Elite 80). A second culture was only irradiated with 2Gy, a third culture was treated with AlCl3.6H2O and not irradiated and a fourth culture received neither treatment. The cells were sampled at 0, 15, 30, 60 and 120 minutes post exposure to the test substance/radiation. The OTM (DNA migration) was used to quantify the DNA damage using at least 50 cells for donor, exposure group and time.


Evaluation criteria:
Alkaline Comet Assay
The Olive Tail Moment calculated by the CASP software was used to quantify DNA damage. Statistical significance and dose-response were considered.

Modified Comet Assay
- The statistical significance and dose-response were considered.
Statistics:
Results were presented as means with standard deviations based on independent experiments conducted using the lymphocytes from two or three donors.

Alkaline Comet Assay
OTM: Differences between the control and treatment groups were evaluated using a one-sided t-test
Apoptosis from morphology (apoptotic index)
One-sided t-test
Apoptosis from flow cytometry
Differences between the control and treatment groups were evaluated using a one-sided t-test.
Other data
- were evaluated using one-way ANOVA followed by the Kruskal-Wallis rank sum test.

The correlation between apoptosis measurements from flow cytometry and morphology (apoptotic index) was conducted using the Pearson product moment.
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
8.3 µM
Vehicle controls validity:
other: It is not clear whether the negative control was a vehicle control or just for conditions.
Untreated negative controls validity:
other: It is not clear whether the negative control was a vehicle control or just for conditions.
Positive controls validity:
not examined
Additional information on results:
Apoptosis
The apoptotic index obtained from the quantification of the number of cells with diffuse fan-shaped tails and small heads was significantly greater than the negative control value at 20.75, 41.5 and 103.75 μM AlCl3. The levels of apoptosis from flow cytometry and fluorescence detection were significantly greater than the negative control at AlCl3 concentrations 8.3 μM and above.

The percent apoptosis at 103.75 μM AlCl3 was close to 20% when assessed using the apoptotic index and greater than 30% using the flow cytometric method. At 20.75 and 41.5 μM the level of apoptosis was between 10 and 20%.

The number of early-stage apoptotic cells was significantly higher than in the negative control at 8.3 μM and above. Early stage apoptotic cells were most prevalent at 41.5 μM and then decreased at 103.75 μM. Late stage apoptotic cells increased with dose exhibiting the highest level at 103.75 μM.

The level of DNA damage assessed by the alkaline Comet Assay increased with dose. At 5 and 10μg/mL AlCl3(20.75 and 41.5μM) the DNA damage was significantly higher than the negative control (p<0.05). At 25μg/mL AlCl3(103.75μM), the level of DNA damaged was lower, not significantly greater than the control. 

 

Oxidative Damage: Modified Comet Assay

The level of oxidised purines and pyrimidines showed an increase with dose, becoming significantly different from the negative control at 8.3μM. Levels peaked at 41.5μM and showed a decrease at 103.75μM.

 

DNA damage in relation to the cell cycle

This data was presented as graphs in the article. In general, the highest levels of DNA damage (OTM) were observed in cells classified as in the S-phase on the basis of their relative DNA content. The variability in the data was high and the results were not statistically significant.

 

Consistent with the other results for initial DNA, the amount of damage increased with dose to 41.5μM and was then lower at 103.75μM.

 

The distribution of cells in the different phases showed some evidence of a cell-cycle delay on treatment with Al with retention of the cells in the S-phase.

 

Kinetics of DNA repair following 2Gy irradiation

Absolute and relative (%) data were presented graphically in the article. On visual examination of the graphs show a return close to control levels of DNA damage by 120 minutes in the irradiated cultures. In the cultures exposed to 41.5μM AlCl3in addition to radiation, the OTM remained elevated at 120 minutes.

Conclusions:
A positive response was observed on exposure of human peripheral lymphocytes to aluminium chloride. A dose response was evident with statistically significant effects compared with the control at 20.75 μM. Apoptosis observed at this test concentration levels was 10-20%. it is unclear whether the increase in the level of DNA damage observed is actually due to oxidative damage.
The results of the study provide some evidence of an increase in the occurrence of oxidised bases with the increase in DNA damage, supporting a role of oxidative damage in the effects observed.
Executive summary:

Lankoff et al. (2006) assessed DNA damage (Olive Tail Moments (OTMs) using CASP software) and apoptosis using the alkaline Comet Assay in human peripheral lymphocytes from three donors who were young (aged 19 to 30) and healthy. Gender and smoking status of the donors were not reported. The test substance was AlCl3.6H2O and the same concentrations were used as in an earlier study by the same group (Banasik et al., 2005): 0, 4.15, 8.3, 20.75, 41.5 and 103.75 µM-Al. Cultures were treated with the test substance for the full 72 hours incubation time.

Apoptosis was estimated by two methods: flow cytometry and the annexin V-FITC apoptosis detection Kit I (BD Pharmingen,) (single donor) and an apoptotic index based on comet shape (i.e. the percent of cells with diffuse fan-tails and small heads among a minimum of 100 cells per donor and dose). Characterization of the distribution of the DNA damage across different phases of the cell cycle was attempted in cells from two donors using the CASP software. The total fluorescence intensity of individual cells was used as an index of relative DNA content and thus of cell-cycle phase of the individual cells. Cells were thus classified as G1, S or G2/M. The results from the alkaline Comet Assay OTM and apoptosis were presented in a graph. The OTM showed an increase with dose becoming significantly greater than the negative control (p<0.05) at AlCl3 concentrations of 20.75 and 41.5 µM-Al. The OTM then decreased at higher concentrations concomitant with a marked increase in apoptosis. Apoptosis measured using flow cytometry was generally higher than that estimated visually using the apoptotic index. At 20.75 and 41.5 µM-Al, levels of apoptosis based on the apoptotic index were about 8 and 10%, respectively. The values from flow cytometry were about 11 and 18%, respectively. At 103.75 µM-Al, apoptosis was about 18% based on the apoptotic index and 32% based on flow cytometry. Oxidative DNA damage assessed using a modified Comet Assay with Endo III (excision of oxidised pyrimidines) and Fpg (excision of oxidised purines) was also reported in this study. Bothoxidised purines and pyrimidines were significantly increased relative to the non-enzyme digested groups at aluminium concentrations of 8.3, 20.75, 41.5 and 103.75 µM-Al. Oxidised purines and pyrimidines showed a similar response profile to overall DNA damage, increasing with dose to 41.5 µM-Al and then decreasing at 103.75 µM-Al. 

Lankoff et al. (2006) also investigated the influence of Al on repair of radiation-induced DNA damage. Prior treatment with 41.5 µM AlCl3 led to a significant decrease in the efficiency of repair of DNA damage induced by gamma radiation (2Gy). The alkaline Comet Assay results from this study were positive but, due to the increase in apoptosis as DNA damaged increased, it is not possible to separate cytotoxic effects from oxidative genotoxic effects based on these results.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: deviation with respect to the number of metaphases scored; lack of details on test substance
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
The human lymphocytes were obtained from heparinized whole blood samples provided by four “ normal, healthy donors”, two men and two women, aged 21 to 26 years with no history of smoking/drinking or chronic drug use.

Medium:
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.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 5, 10, 15 and 25 μM AlCl3.
Vehicle / solvent:
Methanol (CAS#: 67-56-1, Merck-Schuchardt Co.)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Doxorubicin
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 5 mL HAM-F10 (78%, heat-inactivated fetal calf serum (20%), phytohemagglutinin-M (2%) and antibiotics 0.01 mg/mL of penicillin, 0.005 mg/mL streptomycin. Cultures were maintained at 37 ºC 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 0.2 mL PHA and AlCl3 and then incubated for 52 hours at 37 ºC 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 37 ºC 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.
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Ancillary Data:

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.

(*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.

Conclusions:
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.
Executive summary:

Lima et al. (2007) determined the frequency of structural chromosome aberrations in human peripheral lymphocytes obtained from 4 young (aged 21 to 26 years), healthy, non-smoking donors on exposure to 5, 10, 15 and 25μM aluminium chloride (AlCl3) at different phases of the cell cycle. For the G1 phase, lymphocytes were treated with a combination of 0.2 mL PHA and AlCl3 and then incubated for 52 hours at 37 ºC 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. As pointed out by an expert reviewer, the cells would be in exponential growth by 69 hours and not synchronised in G2. The study was generally well-reported but lacked detail in some areas, e.g. the purity of the test compound. It appeared to follow GLP. The number of metaphases assessed was less than that recommended in OECD TG#473 and it is unclear how many cultures were used per dose and time point. The CA results are presented as single values, although results ought to have been available from 4 cultures, i.e. one from each of the 4 donors. No measure of the variability was provided. There is also uncertainty concerning the types of structural aberrations included in the “total” damage index. Both gaps and breaks were included in the index and whether the breaks were chromatid and/or chromosome was not specified. Methanol was used as the vehicle although this does not appear to have compromised the chromosome aberration results from the study. In the presence of metabolic activation, methanol may be metabolised to formaldehye (a DNA cross-linking agent) that can cause a wide range of DNA damage. As metabolic activation was not used in this study, the formation of formaldehyde would be low and the use of methanol as a vehicle is unlikely to have compromised the results of the study. The levels of CA in control cultures were normal, in the range expected for healthy human blood donors, and so it does not appear that the use of methanol compromised the study. Total aberrations (gaps plus breaks) were significantly higher than the control in all treated cultures in the G1 and G1/S phases, the S phase and the G2 phase.

During the G1 phase, the treated cultures also exhibited significantly increased polyploidy and endoreduplication compared with the negative control. Although not reaching statistical significance, breaks alone showed a dose-related increase. The results are positive but require qualification due to reduction of the mitotic index below 50% of the negative control at most of the AlCl3 concentrations tested. The result for chromosome aberrations is positive, but of unclear biological relevance [Klimisch Score=2]. The authors suggested that AlCl3-related adverse effects result from interference with the mitotic apparatus.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
: lack of details on test substance
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Line/Source of Cells:
TK+/- -3.7.2 heterozygote of L5178Y mouse lymphoma cells.

Medium:
Fischer’s medium for leukemic cells mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G, streptomycin sulfate.
Metabolic activation:
without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
570, 580, 590, 600 and 625 µg/mL
Vehicle / solvent:
Sterile, deionized, glass-distilled water
Untreated negative controls:
other: Sterile, deionized, glass-distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: EMS (ethyl methanesulphonate) and AAF ( N-2-fluorenylacetamide (2-acetylaminofluorene))
Details on test system and experimental conditions:
Cell culture processing & conditions:
- cells were thawed from frozen stock, maintained in Fischer’s medium for leukemic cells from mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G, streptomycin sulphate.
- background spontaneous TK-/- mutant frequencies were reduced by weekly 24 hours treatment with medium containing thymidine, hypoxanthine, methotrexate and glycine.

Preparation of test solutions:
Metal compounds diluted in sterile, glass distilled water; 0.1 mL of dilution added to culture medium.

Administration of test solutions:
Volume: 0.1 mL

Duration of exposure, schedule/duration of incubations:
Exposure Duration: 4 hours at 37 ºC.
Schedule: 2-day expression period after washing.

- test solutions were added to a 10 mL suspension containing 6x 106 cells from culture recently treated to remove TK-/-.
- after exposure, cells were washed twice, fresh medium added, cultures carried through a 2-day expression period.

Incubations per dose/time point:
2 or more

Metabolic activation:
10% dilution of S9 in medium; added with cofactor mix containing NADP at 8 mg/mL and isocitric acid (at 15 mg/mL.

Measurement of study outcomes:
- On Day 2: a modified cloning procedure was used. The culture was centrifuged, cells re-suspended at 500,000 viable cells per mL Fischer’s and serial dilutions plated in triplicate in cloning medium with and without trifluorothymidine (TFT). Incubation at 37ºC for ca. 12 days occurred before before colony counting (New Brunswick Scientific automatic colony counter).

Ancillary endpoints examined:
Cell viability was determined by trypan blue dye exclusion.
Total survival: by the method of Clive and Spencer (1975) – combining growth in suspension culture and soft cloning efficiency data.
Evaluation criteria:
Mutant frequency (MF): number of mutants per 105 colony forming cells.

Positive response definition:
“a test agent will be considered positive in the L5178Y mouse lymphoma assay if a dose-related response is obtained in which two or more concentrations elicit a greater then 2-fold increase in MF over the solvent with a minimum of 10% survival (Clive et al., 1979).”
Statistics:
No data.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was a large change in pH when AlCl3 was added to the medium with possible effects on growth.


COMPARISON WITH HISTORICAL CONTROL DATA: values for the positive and solvent controls fell within the range of historical controls for the laboratory.


ADDITIONAL INFORMATION ON CYTOTOXICITY: total survival was not linearly related to dose; subsequent tests with AlCl3 (not described further) resulted in a nonlinear toxic response with little to no increase in mutation frequency.

Results for the other chloride salts provide evidence for the lack of a mutagenic effect of the chloride counter ion at the concentrations of AlCl3 administered.

The mutation frequency (MF) for the solvent was 2.5 with a total survival of 100%. The positive control (EMS) exhibited a mutation frequency of 85 with a total survival of 31%.

 

The MF for AlCl3was constant at approximately 2-times background for 625, 600, 590, 580, and 570 μg/mL. The percentages for total survival at these concentrations were 63, 38, 42, 88 and 69, respectively.

Conclusions:
The negative results for forward mutations require qualification due to the occurrence of pH effects and also due to the failure to reach required levels of cytotoxicity (relative survival 10-20%) at the highest concentration.
Executive summary:

Oberly et al. (1982) did not observe forward mutations at the thymidine kinase (tk) locus in the L5178Y mouse lymphoma assay with the use of AlCl3 at concentrations from 2.36-2.59 mM. The study was well-described and used a standard assay but did not test a suitable range of concentrations. At least 4 analysable concentrations should be used with the maximum resulting in 10-20% relative survival (OECD TG#476, 1997). The lowest relative survival observed in the study was 38% at 600 µg/mL. A further caveat on the reliability of the results for AlCl3 is the observation of a large pH change on addition of this substance to the medium, a change that may influence growth. 

The authors report qualitatively that tests with AlCl3 showed a nonlinear toxic response with, however, little to no increase in mutation frequency. The authors also state that “Other test systems and refinement of test conditions in the mouse lymphoma assay are needed to properly assess the mutagenic potential of this metal”. The results of the study were negative but require qualification as discussed above. It was given a Klimisch Score of 2 but is not considered of adequate quality and reliability to meet the Annex X REACH information requirements for a gene mutation assay in mammalian cells.

Genetic toxicity in vivo

Description of key information

Cytogenicity/chromosome aberration: negative (for non-nano-sized test material)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
5 Jan 2010 – 4 Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: range-finder experiment: 6-10 weeks; micronucleus experiment: 8 weeks
- Weight at study initiation: range-finder experiment: 181-190 g; micronucleus experiment: 217-260 g
- Housing: The rats were housed in an air-conditioned room (15 air exchanges/hour) in groups, up to six per group, “in cages with the 'Code of practice for the housing and care of animals used in scientific procedures”
- Diet (e.g. ad libitum): ad libitum access to SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham)
- Water (e.g. ad libitum): Mains water ad libitum via water bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): fluorescent lighting, 12/12-h light/dark cycle (light from 06:00 to 18:00h)

- Identification: individually, by uniquely numbered ear-tag.
Cages were identified by study number, study type, start date, number and sex of animals, dose level and proposed time of necropsy using a color-coded procedure
Route of administration:
oral: gavage
Vehicle:
1% Carboxymethylcellulose in deionised water (1% CMC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Freshly prepared. As no storage instructions were available, after preparation the formulations were held at 15-25 °C in a dark place and used within 2 hours.

Formulations were mixed using a Silverson homogenizer until visibly homogenous; dose bottles were stirred continuously on a magnetic stirrer before and throughout dosing


Duration of treatment / exposure:
not applicable
Frequency of treatment:
Two doses ≈ 24 hours apart
Post exposure period:
24 hours after the second (final) administration
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Range-finder experiment
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Micronucleus experiment
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Micronucleus experiment
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Micronucleus experiment
No. of animals per sex per dose:
Range-finder experiment – 6 (3 males and 3 females)
Micronucleus experiment – 6 (males only)
Control animals:
yes, concurrent vehicle
Positive control(s):
Substance: Cyclophosphamide (CPA), Sigma-Aldrich Chemical Co, Poole, UK, freshly prepared in saline
Administration: once via oral gavage 24 hours prior to necropsy (dose 20 mg/kg)
Tissues and cell types examined:
Bone marrow cells were obtained from the femur.
Details of tissue and slide preparation:
Measurement of study outcomes
Bone marrow cells were obtained from the femur. Slides were stained with acridine orange and scored using fluorescence microscopy.

“Slides from the CPA-treated rats were initially checked to ensure the system was operating satisfactorily.”

Slides from all groups were arranged by randomly allocated animal number and analyzed by an individual unaware of the animals’ dose group.

The relative proportion of polychromatic erythrocytes (%PCE) was determined by analyzing at least 1000 cells - polychromatic plus normochromatic erythrocytes (NCE)

Frequency of micronucleated PCE (% MN PCE) was determined by analysis for micronuclei (MN) of at least 2000 PCE per animal.

The following data are presented in a tabular form for each animal: PCE and NCE counts; %PCE; micronucleated PCE (MN PCE) per 2000 PCE; %MN PCE

For each group, the following values are presented: cell total, %PCE, total MN PCE, mean MN PCE per 2000 PCE, % MN PCE (SD)

The laboratory historical vehicle control ranges are presented and the MN PCE data from the vehicle control group are compared with these historical data

The laboratory historical positive control ranges are also presented

Ancillary endpoints examined (e.g. general toxicity):
Routine health status checks – at the beginning and the end of each work day.
Range-finder experiment:
- clinical signs of toxicity (immediately after each dose administration, at least 4 times during the four-hour post-administration period and prior to the second dose),
- body weight (each day of dosing and each day post-administration)
- core body temperature (once in the 24 hours pre-administration, 2 and 4 hours after each administration and once on the first post-administration day)
Micronucleus experiment:
- clinical signs of toxicity (immediately after each dose administration, at least 4 times during the 4-hour post-administration period, prior to the second dose and on the day of bone marrow sampling)
- body weight - on the day of bone marrow sampling
- as no changes in body temperature were observed in the range-finder experiment, body temperature was not measured.
Evaluation criteria:
The criteria used for a positive response are provided explicitly.
For the test article to be considered positive (inducing clastogenic/aneugenic damage), all of the following 4 criteria are to be met:
“1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. The group mean MN PCE value at such a point exceeds the 95% calculated confidence interval for the mean historical vehicle control data
4. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).”
If none of the 4 criteria are met, the test article is to be considered negative in this assay.
Results only partially satisfying the above criteria are to be considered on a case-by-case basis. Biological relevance is to be taken into account (e.g. consistency of response within and between dose levels)
Statistics:
Heterogeneity chi-square test was used for evaluation of inter-individual variation in the numbers of MN PCE for each group.

A 2x2 contingency table and chi-square test was used to compare the numbers of MN PCE in each treated group with the numbers in vehicle control groups

A test for linear trend was used to evaluate possible dose-response relationship.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

OECD TG #474: Principal endpoint = Frequency of micronucleated immature (polychromatic) erythrocytes

 

1. The frequency and distribution of MN PCE in the vehicle control group weresimilar to the historical vehicle control data.

2. There was a significant increase in the frequency of MN PCE (% MN PCE) in the positive control group

3. There was no evidence of test-substance-induced bone marrow toxicity, i.e. no decrease in the relative proportions of PCE (%PCE) compared to the vehicle control group and no dose-dependent decrease in %PCE; %PCE in the treated groups were even slightly higher than in the non-treated groups.

4. Group mean frequencies of MN PCE (% MN PCE) in all three dose groups were similar to and not significantly different from those in the vehicle control group.

5. Individual %MN PCE for all treated animals were within the range of historical vehicle control distribution data and similar to those observed in recent historical controls.

------------------------------------------------------------------------

Group      %PCE   MN PCE/2000 PCE    %MN PCE (SD)

(dose)                                                           

-------------------------------------------------------------------------

0              49.35             2.67                   0.13 (0.10)

500           58.55             2.33                   0.12 (0.09)

1000         53.08             2.83                   0.14 (0.09)

2000         54.82             2.50                   0.13 (0.06)

PC*          46.13            55.50                  2.78 (1.60)

-------------------------------------------------------------------------

*PC-positive control

Conclusions:
Aluminium hydroxide adminaterede“It is concluded that aluminium hydroxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 2000 mg/kg/day (the maximum recommended dose for this study)”
Executive summary:

Covance (2010a) administered aluminium hydroxide to out-bred male Sprague Dawley rats to examine the induction of micronuclei (MN) in bone marrow polychromatic erythrocytes (PCE). The animals were randomized into 5 groups (6 animals in each). Three groups were exposed to doses of 500, 1000, and 2000 mg/kg/day, one group (negative control) received the vehicle (1% carboxymethylcellulose in deionised water), and one group (positive control) received a known mutagen, Cyclophosphamide. The test substance was administered by oral gavage in two doses 24 hours apart. The maximum dose tested was selected based on data from a range-finder experiment. The principal endpoint was the frequency of micronucleated PCE (% MN PCE) in the bone marrow, sampled 24 hours after the final test substance administration. The results of the study were negative: group mean % MN-PCE values in all three dose groups were not significantly different from those in the vehicle control group; individual %MN PCE for all treated animals were also within the range of historical vehicle control distribution data.

No signs of general toxicity or bone marrow toxicity (based on the proportions of immature erythrocytes) were observed in this study. The authors concluded: “……aluminium hydroxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 2000 mg/kg/day.” This GLP-compliant study was conducted in accordance with OECD Test Guideline #474 (1997) and European Agency for the Evaluation of Medicinal Products (1995) guidelines. Small deviations were unlikely to impact the validity of the results. A Klimisch Score of 1 was assigned to this study. The MN assay results are reliable but require discussion in the context of toxicokinetic information as Al levels were not determined in the target tissues.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: W NIN (National Institute of Nutrition, Hyderabad, India).
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 90-100 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Diet: ad libitum. The feed was described as standard laboratory feed (wheat flour 2.5%; roasted Bengal gram flour 60%; skimmed milk powder 5%; casein 4%; refined ground oil 4%; salt mixture 4%; vitamin mixture 0.5%).
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture
- Injected Volume: Unclear
- Injected concentration: Unclear as volume was not provided.
- Amount of vehicle (if gavage or dermal): The amount of test substance injected was 47.5 mg, 95 mg and 190 mg Al2O3, equivalent to 25 mg, 50 mg and 100 mg of Al (calculated from the numbers provided in the article).

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Al2O3 particles were suspended in 1% Tween 80 (a surfactant that enhances uptake), dispersed by ultrasonic vibration for 10 minutes and “mixed thoroughly” prior to use.
Duration of treatment / exposure:
Genotoxicity Assays
Exposure Duration: Single exposure
Schedule/Frequency of Administrations: Single, acute
Frequency of treatment:
Not applicable.
Post exposure period:
CA assay: 18h, 24 h

Mitotic Index (MI): 18h, 24h

Al Level Study:
Urine and faeces: 48 h after dosing
Blood and tissues: 14 days after dosing??
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amount of 47.5 mg Al2O3 assuming a body weight of 95 g or 25 mg Al
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amount of 95 mg Al2O3 assuming a body weight of 95 g or 50 mg Al
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amount of 95 mg Al2O3 assuming a body weight of 95 g or 100 mg Al
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Substance: Cyclophosphamide
Justification: Known mutagen on list of acceptable positive control substances in the relevant OECD TGs.
Route of administration(s): intraperitoneal
Dose/concentration: 40 mg/kg bw, volume = 0.01 mL/g bw
Tissues and cell types examined:
Rat bone marrow.
Details of tissue and slide preparation:
CA Assay:
The authors stated that the measurements were made in accordance with OECD TG#475. 500 well-spread metaphases (100 per animal) were analyzed for each treatment (dose/time). Aneuploidy, polyploidy, gaps, breaks, minutes, acentric fragments and reciprocal translocations were counted.

Al-Levels:
0.1 - 0.3 g of fresh tissue were predigested in ultrapure nitric acid overnight, heated to 80 ºC for 10 h and then 130 - 150 ºC for 30 minutes. The samples were then heated (temperature not provided) for an additional 4 hours in the presence of 0.5 mL 70% perchloric acid and evaporated to dryness. Solutions were then made up to 5 mL with deionized water, filtered and the Al concentration was determined using ICP-MS with rhodium at 20 ng/mL as an internal standard.

Cytotoxicity (Mitotic Index)
The MI was determined on 1000 cells or more from randomly selected slides that were coded prior to scoring.
Evaluation criteria:
According to guideline.
Statistics:
The methods used were included in the article but were not well-described. The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. Testing for homogeneity of variances was also not mentioned. The results show some evidence of an increase in the magnitude of the standard deviation with dose for some endpoints.
Sex:
female
Genotoxicity:
ambiguous
Remarks:
CA Assay: Al2O3 - (50-200 μm)
Toxicity:
no effects
Remarks:
The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
positive
Remarks:
CA Assay: Al2O3 - (30nm)
Toxicity:
no effects
Remarks:
The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
positive
Remarks:
CA Assay: Al2O3 - (40nm)
Toxicity:
no effects
Remarks:
The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
MI: There was no significant reduction in MI at either sampling time for any of the dose groups compared with the vehicle control.

At 18h:
Negative control: 2.97(±0.12)
Treated groups: The MI ranged from 91 to 110% of the value in the negative control.
Positive control: 75% of negative control
At 24h:
Negative control: 3.25±0.10 (mean, sd)
Treated groups: The MI ranged from 82 to 90% of the value in the negative control.
Positive control: 80% of negative control

General toxicity: The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.

CA assay:

Total aberrations (excluding gaps)

 

Negative control (1% Tween 80), mean (±sd)

18h: 0.6(±0.32)

24h: 0.5(±0.42)

 

Al2O3- (50-200 μm)

18h:

25 mg Al:0.6±0.22 - ns

50 mg Al: 2.2±0.79 - ns

100 mg Al: 4.3±1.01 - ns

(increase evident with dose but not reported as statistically significant)

24h:

25 mg Al:0.5±0.31 - ns

50 mg Al: 1.2±0.51 - ns

100 mg Al: 5.5±0.31 - ns

(increase evident with dose but not reported as statistically significant)

Al2O3- (30nm)

18h

25 mg Al: 2.3±0.52 - ns

50 mg Al: 6.9±0.61 - p<0.05

100 mg Al: 11.8±1.10 - p<0.001

24h

25 mg Al: 1.2±0.23 - ns

50 mg Al: 7.3±0.59 - p<0.05

100 mg Al: 10.5±1.31 - p<0.001

Al2O3- (40nm)

18h

25 mg Al: 1.8±0.36 - ns

50 mg Al: 4.5±0.53 - ns

100 mg Al: 9.6±1.84 - p<0.001

24h

25 mg Al: 1.2±0.29 - ns

50 mg Al: 4.3±0.37 - ns

100 mg Al: 9.9±1.05 - p<0.001

- No polyploidy or reciprocal translocations observed in the Al-treated groups;

- Aneuploidy: clear dose response (not statistically tested for trend) observed for the 30 nm and 40 nm groups with significantly higher numbers in the highest dose group (p<0.001).  Al-levels: “Al-content”μgAl2O3/g wet tissue:

Control (units±sd)

Whole blood   3±1 μg/g

Liver              6±2μg/g

Spleen            2±1μg/g

Heart             6±3 μg/g

Kidneys          9±4 μg/g

Brain              2±3 μg/g

Urine (48 hours post-dosing) 5±2 μg/mL

Faeces (48 hours post-dosing) 1±1 mg/g

 

Al2O3- (50-200 μm)”bulk”

The levels in tissues show an increase with dose that does not reach statistical significance. A significant (p<0.01) increase in the Al2O3content in faeces relative to the control was observed at 2000 mg/kg bw.

 

Al2O3- (30nm, 40nm)

There is considerable variability in the results, but all tissue concentrations in the 30nm and 40nm treatment groups show an increase with dose; pairwise comparisons between treatment and controls groups are reported as statistically significant. The highest levels were observed in the kidneys and brain.

Conclusions:
The results were positive for the nano-sized materials with evidence of a positive dose-response relationship for CAs.
The results were positive for the nano-sized materials with evidence of a positive dose-response relationship for CAs. The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those of the vehicle control. The positive results for the nano-sized materials may have resulted, in part, from the nanoparticles as foreign bodies in the bone marrow as opposed to effects from the chemical species (complexed “Al3+”) itself. Current scientific knowledge does not allow the separation of these effects. The relevance of the effects observed for the nano-sized particles to the target compounds in the specified exposure scenarios is unclear.

Aluminium (oxide) levels were elevated in all tissues in a dose-response manner after dosing with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The reported levels of Al in the urine of the control group were three orders of magnitude greater than “normal” levels in humans.
Executive summary:

Balasubramanyam et al. (2009a) administered suspensions of aluminium oxide by oral gavage to female albino Wistar rats (5 animals per group). Concentrations of 500, 1000 and 2000 mg l2O3/kg bw in 1% Tween 80/doubly-distilled water (DDW) were administered to the rats. These concentrations are equivalent to 265, 529 and 1058 mg Al/kg bw. Three size fractions of aluminium oxide particles were examined: Al2O3 (30 nm; transmission electron microscopy (TEM) determined diameter, mean±sd - 39.85±31.33 nm), Al2O3 (40 nm; TEM diameter, mean±sd – 7.33±36.13 nm), and Al2O3-bulk (diameter 50 to 200 μm). A negative control group was treated orally with the Tween 80/DDW vehicle. A positive control group received a single intraperitoneal dose of 40 mg/kg bw of cyclophosphamide. The assessment of CA in bone marrow cells was conducted in accordance with OECD Test Guideline #475 with the analysis of 500 well-spread metaphases (100 per animal) for each treatment 18 and 24 hours after the last dosing. Aneuploidy, polyploidy, gaps, breaks, minutes, acentric fragments and reciprocal translocations were counted. The mitotic index was determined on 1000 cells at both sampling times, slides were selected randomly and coded to blind analysts. Dose levels were determined using an acute oral toxicity study conducted in accordance with OECD Test Guideline #420. Organ tissue was analyzed for aluminium content by ICP-MS (inductively coupled mass spectrometry). The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. There was no indication of an effect of treatment on the mitotic index. 

Eighteen hours after the final dosing, total chromosome aberrations (including and excluding gaps) were significantly higher than in the control for both the 529 mg Al/kg bw (p<0.05) and 1058 mg Al/kg bw (p<0.001) dose groups in cells from the Al2O3 (30 nm) nanomaterial group. For the Al2O3 (40 nm) group, total chromosome aberrations were significantly higher than the control only in cells from the 1058 mg Al/kg bw dose group (p<0.001). For the individual types of chromosome aberrations, significant differences for pairwise comparisons between treatment groups and control were observed only for aneuploidy. Gaps, breaks, minutes and acentric fragments showed some evidence of increases with dose in the Al2O3 (30 nm) and Al2O3 (40 nm) groups, however. Statistical testing reported in the article indicated no significant differences for any treatment level of Al2O3-bulk compared with the control group. The mean±sd total aberrations for the control, 265, 529 and 1058 mg Al/kg bw/day Al2O3-bulk groups were 0.6±0.3, 0.6±0.3, 2.2±0.8, and 4.3±1.0, respectively. This study used only one gender of animal but appears to have been well-conducted although some elements of reporting are lacking. A Klimisch Score of 2 is considered appropriate for the CA results.

Balasubramanyam et al. (2009a) also reported measurements of levels of Al in the urine and faeces sampled 48 hours after dosing and in tissues in samples taken 14 days after dosing. The table showing the tissue analysis results was almost identical to that in Balasubramanyam et al. (2009b) with the exception that the tissue values were reported as “Al content” in contrast to “Al2O3 content” in Balasubramanyam et al. (2009b). Contact with the author clarified that the units used in the table for tissue doses were μg Al2O3/g wet tissue. Aluminium levels were elevated in all tissues in a dose-response manner with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The measurements do, however, show consistent increases inlevels of Al in tissues and organs with increasing dose. Nano-sized materials were absorbed to a greater extent than Al2O3-bulk (50-200µm). The units used in the article for levels in urine (µg/mL) indicate Al concentrations in the urine of the control group to be 3-orders of magnitude higher than the normal range in humans (1-2 µg/L) (Krewski et al., 2007). The reliability of the aluminium measurements in tissues and urine is limited by the inconsistencies and lack of clarity in reporting. A Klimisch Score of 3 was assigned to the Al tissue measurements.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: W NIN (National Institute of Nutrition, Hyderabad, India).
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 – 100 g
- Diet: ad libitum. The feed was described as standard laboratory feed (wheat flour 2.5%; roasted Bengal gram flour 60%; skimmed milk powder 5%; casein 4%; refined ground oil 4%; salt mixture 4%; vitamin mixture 0.5%).
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture
- Injected Volume: Unclear
- Injected concentration: Unclear as volume was not provided.
- Amount of vehicle (if gavage or dermal): The amount of test substance injected was 47.5 mg, 95 mg and 190 mg Al2O3, equivalent to 25 mg, 50 mg and 100 mg of Al (calculated from the numbers provided in the article).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Al2O3 particles were suspended in 1% Tween 80 (a surfactant that enhances uptake), dispersed by ultrasonic vibration for 10 minutes and “mixed thoroughly” prior to use.
Duration of treatment / exposure:
Genotoxicity Assays
Exposure Duration: Single exposure
Schedule/Frequency of Administrations: Single, acute
Frequency of treatment:
Not applicable.
Post exposure period:
MN assay: 30h, 48h

Mitotic Index (MI): 18h, 24h

Al Level Study:
Urine and faeces: 48 h after dosing
Blood and tissues: 14 days after dosing??
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amounts of 47.5 mg Al2O3 assuming a body weight of 95 g or 25 mg Al
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amounts of 95 mg Al2O3 assuming a body weight of 95 g or 50 mg Al
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amounts of 190 mg Al2O3 assuming a body weight of 95 g or 100 mg Al
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Substance: Cyclophosphamide
Justification: Known mutagen on list of acceptable positive control substances in the relevant OECD TGs.
Route of administration(s): intraperitoneal
Dose/concentration: 40 mg/kg bw, volume= 0.01mL/g bw
Tissues and cell types examined:
Rat bone marrow.
Details of tissue and slide preparation:
MN assay:
The authors stated that the measurements were made in accordance with OECD TG#474. A minimum of 4 slides were made for each animal for assessment of MN frequency and stained with Giemsa. Slides were coded. A total of 2000 PCEs (polychromatic erythrocytes) from all 4 slides were scored for MN.

Al-Levels:
0.1-0.3g of fresh tissue were predigested in ultrapure nitric acid overnight, heated to 80ºC for 10h and then 130-150ºC for 30 minutes. The samples were then heated (temperature not provided) for an additional 4 hours in the presence of 0.5mL 70% perchloric acid and evaporated to dryness. Solutions were then made up to 5mL with deionized water, filtered and the Al concentration was determined using ICP-MS with rhodium at 20ng/mL as an internal standard.

Cytotoxicity (Mitotic Index):
The MI was determined on 1000 cells or more from randomly selected slides that were coded prior to scoring.
Evaluation criteria:
The criteria used for a positive response were not provided explicitly but the guidelines were cited.

OECD TG #474: reports that there are several criteria for determining a positive result e.g. dose-related increase or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. “Biological relevance of the results should be considered first.”

Statistics:
The methods used were included in the article but were not well-described. The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. Testing for homogeneity of variances was also not mentioned. The results show some evidence of an increase in the magnitude of the standard deviation with dose for some endpoints.
Sex:
female
Genotoxicity:
negative
Remarks:
MN-Assay: Al2O3 - (50-200 μm)
Toxicity:
no effects
Remarks:
The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
positive
Remarks:
MN-Assay: Al2O3 - (30nm)
Toxicity:
no effects
Remarks:
The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
positive
Remarks:
MN-Assay: Al2O3 - (40nm)
Toxicity:
no effects
Remarks:
The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
MI: There was no significant reduction in MI at either sampling time for any of the dose groups compared with the vehicle control.

At 18h:
Negative control: 2.97(±0.12)
Treated groups: The MI ranged from 91 to 110% of the value in the negative control.
Positive control: 75% of negative control
At 24h:
Negative control: 3.25±0.10 (mean, sd)
Treated groups: The MI ranged from 82 to 90% of the value in the negative control.
Positive control: 80% of negative control

General toxicity: The authors briefly mention that no mortality nor toxic symptoms were observed at any dose level in the range-finding study (OECD TG #420) nor in the 5 rats at the highest dose level in the main study that was reported in the article.

MN Assay:

OECD TG #474: Principal endpoint = Frequency of micronucleated immature (polychromatic) erythrocytes

 

The results of the ANOVA omnibus test for a difference between groups was not provided.

None of the treated groups had significantly lower %PCEs compared with the control group.

 

Negative control (1% Tween 80)

MN-PCEs/2000 PCEs, mean ±sd

30h: 2.5 ±0.70

48h: 1.8 ±0.75

Al2O3- (50-200 μm)

30h and 48 h: The frequency of MN-PCEs increased with dose but the pair-wise comparisons with the negative control were not significant. The standard deviation of the results (based on 5 animals per dose) showed an increase with dose.

MN-frequencies at 30h and 48h were similar.

30h:

25 mg Al: 1.9±0.73, ns

50 mg Al: 3.3±1.16, ns

100 mg Al: 5.9±1.71, ns

48h:

25 mg Al: 2.0±0.64, ns

50 mg Al: 4.2±1.07, ns

100 mg Al: 6.6±1.68, ns

 

Al2O3- (30nm)

30h:

25 mg Al: 4.3±1.03, ns

50 mg Al: 9.4±1.87, p<0.01

100 mg Al: 15.2±2.3, p<0.001

48h:

25 mg Al: 5.0±1.05, ns

50 mg Al: 10.6±1.68, p<0.01

100 mg Al: 16.6±2.66, p<0.001

MN-frequencies at 30h and 48h were similar.

Al2O3- (40nm)

30h:

25 mg Al: 3.8±1.05, ns

50 mg Al: 8.1±1.80, p<0.05

100 mg Al: 13.9±2.21, p<0.001

48h:

25 mg Al: 3.9±1.08, ns

50 mg Al: 9.0±1.38, p<0.05

100 mg Al: 14.7±1.68, p<0.001

MN-frequencies at 30h and 48h were similar.

Conclusions:
The results were positive for the nano-sized materials with evidence of a positive dose-response relationship for MN.
The results were positive for the nano-sized materials with evidence of a positive dose-response relationship for MN. The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those of the vehicle control. The positive results for the nano-sized materials may have resulted, in part, from the nanoparticles as foreign bodies in the bone marrow as opposed to effects from the chemical species (complexed “Al3+”) itself. Current scientific knowledge does not allow the separation of these effects. The relevance of the effects observed for the nano-sized particles to the target compounds in the specified exposure scenarios is unclear.

Aluminium (oxide) levels were elevated in all tissues in a dose-response manner after dosing with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The reported levels of Al in the urine of the control group were three orders of magnitude greater than “normal” levels in humans.
Executive summary:

Balasubramanyam et al. (2009a) also determined the frequency of micronuclei in polychromatic (immature) erythrocytes (MN-PCEs) in bone marrow according to OECD Test Guideline #474 (1997). The percentage of PCEs was not significantly different from the vehicle control (1% Tween 80 in DDW) in any treated group indicating that cell death was not occurring as a result of treatment. At both 30 and 48 hours after administration of the last dose, both the Al2O3 (30 nm) and Al2O3 (40 nm) groups showed evidence of a positive dose response for the number of MN-PCEs. At 48 hours, the number of MN-PCEs per 2000 PCEs scored was 1.8±0.75 in the negative control group,5.0±1.1 in the 265 mg Al/kg bw/day group, 10.6±1.7 in the 529 mg Al/kg bw/day group and 16.6±2.7 in the 1058 mg Al/kg bw/day group for Al2O3 (30 nm). The value for the cyclophosphamide positive control was 30.2±4.2. The results were positive for the nanoparticles but negative for the 50 to 200 µm sized Al2O3-bulk particles. A Klimisch Score of 2 was assigned to the MN assay results from this study.

Balasubramanyam et al. (2009a) also reported measurements of levels of Al in the urine and faeces sampled 48 hours after dosing and in tissues in samples taken 14 days after dosing. The table showing the tissue analysis results was almost identical to that in Balasubramanyam et al. (2009b) with the exception that the tissue values were reported as “Al content” in contrast to “Al2O3 content” in Balasubramanyam et al. (2009b). Contact with the author clarified that the units used in the table for tissue doses were μg Al2O3/g wet tissue. Aluminium levels were elevated in all tissues in a dose-response manner with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The measurements do, however, show consistent increases inlevels of Al in tissues and organs with increasing dose. Nano-sized materials were absorbed to a greater extent than Al2O3-bulk (50-200µm). The units used in the article for levels in urine (µg/mL) indicate Al concentrations in the urine of the control group to be 3-orders of magnitude higher than the normal range in humans (1-2 µg/L) (Krewski et al., 2007). The reliability of the aluminium measurements in tissues and urine is limited by the inconsistencies and lack of clarity in reporting. A Klimisch Score of 3 was assigned to the Al tissue measurements.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: W NIN (National Institute of Nutrition, Hyderabad, India).
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 – 120 g
- Diet: ad libitum. The feed was not described (from the companion article – the feed was standard laboratory feed wheat flour 2.5%; roasted Bengal gram flour 60%; skimmed milk powder 5%; casein 4%; refined ground oil 4%; salt mixture 4%; vitamin mixture 0.5%).
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 60 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture or Tween 80 (1%) in phosphate buffered saline
- Injected Volume: 1 mL per 100 g bw
- Injected concentration: approximately 25 g, 50 g and 100 g/L for the three dose levels.
- Amount of vehicle (if gavage or dermal): The amount of test substance injected was 47.5 mg, 95 mg and 190 mg Al2O3, equivalent to approximately 25 mg, 50 mg and 100 mg of Al (calculated from the numbers provided in the article).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Al2O3 particles were suspended in 1% Tween 80 (a surfactant that enhances uptake), dispersed by ultrasonic vibration for 10 minutes and “mixed thoroughly” prior to use.
Duration of treatment / exposure:
Exposure Duration: Single exposure

Frequency of treatment:
Schedule/Frequency of Administrations: Single, acute
Post exposure period:
MN assay: 48h, 72h

Cell viability (trypan blue exclusion assay): not reported further

Al Level Study:
Urine and faeces: 48 h after dosing
Blood and tissues: 14 days after dosing
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amounts of 47.5 mg Al2O3 assuming a body weight of ca. 100 g eqivalent to 25 mg Al
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amounts of 95 mg Al2O3 assuming a body weight of ca. 100 g eqivalent to 50 mg Al
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amounts of 190 mg Al2O3 assuming a body weight of ca. 100 g eqivalent to 100 mg Al
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
Route of administration(s): intraperitoneal
Dose/concentration: 40 mg/kg bw, volume= 0.01 mL/g bw
Tissues and cell types examined:
Rat bone marrow.
Details of tissue and slide preparation:
MN assay:
The authors stated that the measurements were made in accordance with OECD TG#474. PCE (polychromatic erythrocyte) frequency was determined using slides stained with Acridine Orange on 1000 erythrocytes per animal. Slides were coded. The number of micronucleated PCEs was determined using 2000 PCEs per animal.

Al-Levels:
0.1 - 0.3 g of fresh tissue were predigested in ultrapure nitric acid overnight, heated to 80 ºC for 10 h and then 130 - 150 ºC for 30 minutes. The samples were then heated (temperature not provided) for an additional 4 hours in the presence of 0.5 mL 70% perchloric acid and evaporated to dryness. Solutions were then made up to 5 mL with deionized water, filtered and the Al concentration was determined using ICP-MS with rhodium at 20 ng/mL as an internal standard.

Cytotoxicity (cell viability):
Trypan blue exclusion assay (no further detail provided)

General toxicity: no detail provided.
Evaluation criteria:
The criteria used for a positive response were not provided explicitly but the guideline and a published article with credible guidance were cited.

Tice et al. (2000): “a concentration-related increase or decrease in DNA migration and a significant corresponding increase or decrease in DNA migration at one or more dose groups.”
Statistics:
Means of individual animals or experimental units were compared by one-way ANOVA with multiple pairwise comparisons done using Tukey’s test. The group means for each experiment were compared using a singe;-sided Student’s t-test. Testing for homogeneity of variances was not mentioned.
Sex:
female
Genotoxicity:
negative
Remarks:
MN-Assay: Al2O3 - (50-200 μm)
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
positive
Remarks:
MN-Assay: Al2O3 - (30nm)
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
positive
Remarks:
MN-Assay: Al2O3 - (40nm)
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity: trypan blue exclusion assay. Results were reported qualitatively that the percentage viability was >80% for all treated groups at all time points for the Comet Assay.

General toxicity: The authors briefly mention the range-finding study conducted in accordance with (OECD TG #420).


MN Assay:

OECD TG #474: Principal endpoint = Frequency of micronucleated immature (polychromatic) erythrocytes

 

The results of the ANOVA omnibus test for a difference between groups was not provided.

 

None of the treated groups had significantly lower %PCEs compared with the control group. At 48 hours, the % PCEs was 3.43 in the negative control, 1.52 in the positive control and ranged from 2.30 to 3.21% in the treated groups.

 

Negative control (1% Tween 80)

MN-PCEs/2000 PCEs, mean ±sd

48h: 1.51 ± 0.93

72h: 1.63 ± 0.83

Al2O3- (50-200 μm)

48h and 72 h: The frequency of MN-PCEs increased minimally with dose but the pair-wise comparisons with the negative control were not significant.

48h:

25 mg Al: 1.75 ± 0.67, ns

50 mg Al: 1.98 ± 0.98, ns

100 mg Al: 3.99 ± 1.29, ns

72h:

25 mg Al: 1.56 ± 0.76, ns

50 mg Al: 2.10 ± 1.12, ns

100 mg Al: 2.40 ± 1.39, ns

MN-frequencies at 48h and 72h were similar.

Al2O3- (30nm)

48h:

25 mg Al: 2.77 ± 1.23, ns

50 mg Al: 8.20 ± 2.17, p<0.05

100 mg Al: 15.81± 3.45, p<0.01

72h:

25 mg Al: 2.89 ± 1.45, ns

50 mg Al: 6.12 ± 1.86, p<0.05

100 mg Al: 9.21± 2.10, p<0.01

Al2O3- (40nm)

48h:

25 mg Al: 2.45 ± 1.56, ns

50 mg Al: 7.51 ± 2.25, p<0.05

100 mg Al: 12.08 ± 3.18, p<0.01

72h:

25 mg Al: 2.67 ± 1.78, ns

50 mg Al: 5.45 ± 1.65, p<0.05

100 mg Al: 8.31 ± 2.47, p<0.01

Al-levels: “Al-content”

Control (units±sd)

Whole blood 3±1 μg/g

Liver 6±2 μg/g

Spleen 2±1 μg/g

Heart 6±3 μg/g

Kidneys 9±4 μg/g

Brain 2±3 μg/g

Urine (48 hours post-dosing) 5±2 μg/mL

Faeces (48 hours post-dosing) 1±1 mg/g

The tissue concentrations in the control group are similar to levels reported in the literature for normal, healthy human subjects in the literature (Priest, 2004).

Al2O3 - (50-200 μm)”bulk”

The results show an increase with dose that does not reach statistical significance.

Al2O3 - (30 nm, 40 nm)

There is considerable variability in the results, but all tissue concentrations in the 30nm and 40nm treatment groups show an increase with dose; pairwise comparisons between treatment and controls groups are reported as statistically significant. The highest levels were observed in the kidneys and brain.

If the results are reported as μg Al/g, brain levels in the groups dosed with nano-sized particulates exceeded those associated with dialysis encephalopathy in humans (Priest, 2004) for the groups treated with a single dose of 50 and 100 mg.

Conclusions:
The results were positive for the nano-sized materials (30 and 40 nm) with evidence of a dose-response relationship for MN.
The results were positive for the nano-sized materials with evidence of a dose-response relationship for MN and for DNA damage assessed using the alkaline Comet Assay. The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those for the vehicle control even for the sensitive alkaline Comet Assay. The positive results for the nano-sized materials may have resulted, in part, from the nanoparticles as foreign bodies in the peripheral erythrocytes as opposed to effects from the chemical species (“Al3+”) itself. Current scientific knowledge does not allow the separation of these effects.

Aluminium levels were elevated in all tissues in a dose-response manner 14 days after dosing with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls.
Executive summary:

Balasubramanyam et al. (2009b) reported results from the MN Assay carried out using peripheral erythrocytes in female albino Wistar rats administered suspensions of aluminium oxide by oral gavage (5 animals per group). Concentrations of 500, 1000 and 2000 mg Al2O3/kg bw in 1% Tween 80/doubly-distilled water (DDW) were administered to the rats. These concentrations are equivalent to 265, 529 and 1058 mg Al/kg bw. Three size fractions of aluminium oxide particles were examined: Al2O3 (30 nm; transmission electron microscopy (TEM) determined diameter, mean±sd - 39.85±31.33 nm), Al2O3 (40 nm; TEM diameter, mean±sd – 47.33±36.13 nm), and Al2O3-bulk (diameter 50 to 200 μm). A negative control group was treated orally with the 1% Tween 80/DDW vehicle. A positive control group received a single intraperitoneal dose of 40 mg/kg bw of cyclophosphamide. Dose levels were determined using an acute oral toxicity study conducted in accordance with OECD Test Guideline #420. Statistical testing was carried out using Tukey’s test, a more conservative test than the LSD test, for the multiple pair-wise comparisons conducted after one-way ANOVA. The authors also indicated using a single-sided Student’s t-test for comparing group mean values for each experiment. The authors referred to the OECD Test Guideline #474 for their MN assay methods. A dose-response relationship was evident for the number of MN-PCEs for both the Al2O3 (30 nm) and Al2O3 (40 nm) treated groups. No statistically significant effect was evident for the larger particulates, Al2O3-bulk. This assay appears to have been conducted in accordance with GLP and the MN assay results are assigned a Klimisch Score of 2.  

 

Balasubramanyam et al. (2009b) also reported measurements of levels of Al the urine, tissues and faeces 48 hours after the dosing. The table showing the tissue analysis results was almost identical to that in Balasubramanyam et al. (2009a) in which it was stated, “……animals were sacrificed after 14 days of acute oral treatment”. In Balasubramanyam et al. (2009b), it was stated that rat tissues were analysed for aluminium content “in animals sacrificed 14 days after a single oral dose”. The tissue values were reported as “Al2O3 content” in contrast to “Al content” as reported in Balasubramnyam et al. (2009a). Contact with the author clarified that the units used in the table for tissue doses were μg Al2O3/g wet tissue. Aluminium levels were elevated in all tissues in a dose-response manner 14 days after dosing with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The reliability of the aluminium measurements in tissues and urine is limited by the inconsistencies in reporting. A Klimisch Score of 3 was assigned to the Al tissue measurements.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro there is only one Ames test of aluminium oxide available, while there are few data available on the genotoxic potential of aluminium oxide in-vivo.

 

Information available on the genotoxic potential of aluminium compounds was taken into account for hazard assessment, since the pathways leading to toxic outcomes are considered to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) (Krewski et al., 2007; ATSDR, 2008). A detailed rationale and justification for the analogue read-across approach is provided in the technical dossier (see IUCLID section 13).

The publications and reports discussed above have been reviewed by an independent genotoxicity expert (Prof. D J Kirkland), who concurs with the summaries, and the overall weight of evidence for genotoxicity discussed below.

Human Studies

Two human studies, Botta et al. (2006) and Iarmarcovai et al. (2005), became available following the completion of the ATSDR (2008) and Krewski et al. (2007) reviews of aluminium. However, these studies contribute little to the weight of evidence approach for assessing the mutagenic potential of the target substances as the results are confounded by the complex nature of the exposure (a mixture of fume from different welding materials), possible co-exposures, and uncertainties concerning the completeness of the adjustments for age and smoking. 

 

Bacterial test systems

Bacterial mutagenicity assays of simple aluminium compounds have been negative in bacterial reverse mutation tests using several strains of S. typhimurium (e.g.; Marzin and Phi, 1985 (TA102); Ahn and Jeffrey, 1994 (TA98); Gava et al., 1989 (TA92, TA98, TA100, TA104); Blevins and Taylor, 1982 (Spot Test: TA98, TA100, TA1535, TA1537, TA1538); Pan et al., 2010 (TA97a, TA100)) and Escherichia coli WP2 trp uvrA (Pan et al., 2010). Results from the Rec Assay using Bacillus subtilis H17 (Rec+, arg-, tryp-) and B. subtilis M45 (Rec-, arg-, tryp-) have also been negative (Nishioka et al., 1975; Kanematsu et al., 1980).  

As uptake of aluminium depends on the chemical form of the aluminium substance, particularly its solubility and other ligands present, the available mutagenicity assays have tended to use aluminium (III) salts that are more soluble than the target substances.  It should be noted that some strains of bacteria are sensitive to only certain types of genetic changes (Battersby et al., 2007) requiring the use of, for example, a range of S. typhimurium strains to provide suitable sensitivity. Strains TA102 and TA104 are sensitive to oxidising mutagens. Given the possibility of false negatives due to the exclusion of Al3+ ion by cell membranes, and possible insensitivity of assays to mechanisms of metal genotoxicity e.g. induction of large DNA deletions which would lead to cell death rather than mutation (see also Battersby et al., 2007), negative results from bacterial test systems, although contributing to the weight of evidence, are not sufficient to negate further testing.

Pan et al. tested the mutagenic potential of the aluminium oxide (Al2O3, nanopowder) in the Bacterial Reverse Mutation Test (Klimisch score = 2). The test method used was similar to OECD Guideline 471 (1997). However, only 3 tester strains were used; the Salmonella typhimurium histidine auxotrophs TA97a and TA100 as well as Escherichia coli WP2 trp uvrA. For metabolic activation the S9 fraction from Aroclor 1254 induced rats was used. The test substance was solved in water. In the experiment using the standard plate incorporation procedure, 4 dose levels from 0 - 1000 µg/plate were plated with overnight cultures of TA 97a, TA 100 and WP2 in the absence of rat S9 mix. For the experiment with metabolic activation the pre-incubation method was used with the same dose levels as for the plate incorporation test. All test plates (with or without S9 activation) were incubated for 72 h at 37 °C before the colonies were counted. There was no indication of bacteriotoxic effects of Al2O3 nanopowder at doses of up to and including 1000 µg/plate. None of the three strains tested showed an increase in mutant counts over those of the vehicle controls, with and without metabolic activation. The respective positive controls caused an increase in the number of revertants which proved the sensitivity of the test. Therefore, under the conditions of this study, no indications of mutagenic effects of Al2O3 could be found at doses up to 1000 µg/plate in any of the S. typhimurium and E. coli strains tested.

 

Animal Studies – In-vivo Somatic Cell Tests

The most relevant and methodologically strongest studies are those conducted by Covance (2010a) and by Balasubramanyam et al. (2009a,b). Covance (2010a) investigated the induction of micronuclei in the bone marrow of rats treated with aluminium hydroxide by oral gavage. This study was conducted in accordance with GLP and recognized testing guidelines (Klimisch Score=1). No induction of micronuclei was observed even at the highest dose administered – 2000 mg Al(OH)3/kg bw/day, two administrations 24 hours apart, equivalent to ca. 690 mg Al/kg bw/day.

Balasubramanyam et al. (2009a,b) examined the genotoxic effects of aluminium oxide particles in vivo. Single doses of aluminium oxide particulate suspensions were administered to rats by oral gavage. The reporting of these investigations was lacking in some areas but the studies appear to have been conducted according to GLP. The study results were positive for the nano-sized materials with evidence of a dose-response relationship. The relevance of these results to the current hazard identification is unclear as it is not distinghuishable if the observed effects have arisen from the presence of nanoparticles rather than from any solubilized chemical species (“Al3+”) or the chemical substance Al2O3 itself. Low toxicity, poorly soluble substances, such as Al2O3, when in the form of nanoparticles, have produced inflammatory effectsin vitro, possibly due to production of reactive oxygen species (ROS) (Duffin et al., 2007; Dey et al., 2008). Current scientific knowledge does not allow the distinguishing of genotoxic effects due to the physical (in this case “nanoparticle”) nature of the exposure from genotoxic effects due to the chemical characteristics of the substance (Landsiedel et al., 2009; Singh et al., 2009; Gonzalez et al., 2008). However, in the current extensive debate concerning the genotoxic effects of nanoparticles of many different substances, the possibility that nanoparticles stimulate an inflammatory response which leads to oxidative stress and hence to DNA damage has been widely voiced. The genotoxicity levels for 50 to 200μm diameter particles (Al2O3-bulk) were also not statistically significantly different from those for the control. Balasubramanyam et al. (2009a,b) reported tissue aluminium oxide levels elevated in a dose-response manner for the groups treated with nano-sized materials, consistent with transfer of the nano-sized particles across the gastrointestinal mucosa (Florence, 1997; Hagens et al., 2007). A particle size dependence of gastrointestinal absorption was apparent. Aluminium oxide levels in the tissues of animals dosed with the larger 50 to 200μm diameter particles (Al2O3-bulk) were not elevated to a statistically significant level. 

The positive results observed in studies of aluminium sulphate reported by Dhir et al. (1990) and Roy et al. (1992) occurred with non-physiologically-relevant intraperitoneal administration of the test substances and were methodologically weaker (Klimisch Scores of 2-3).

Turkez et al. (2010) studied the clastogenic activity of AlCl3 in hepatocytes of adult male Sprague–Dawley rats (8-weeks old, 5 animals per group) after gavage with 34 mg/kg bw AlCl3 and along with 50 mg/kg bw propolis for 30 days. Isolated hepatocytes were prepared by the collagenase perfusion technique (Wang et al., 2002). Isolated hepatocytes were stained with orange (AO)–40,6-diamidino-2-phenylindole dihydrochloride (DAPI) (AO, 0.5 mg/mL; DAPI, 10 mg/mL) and the numbers of micronucleated hepatocytes (MNHEPs) were counted in 2000 hepatocytes for each animal. MNHEPs were defined as hepatocytes with round or distinct MNs that stained like the nucleus, with a diameter 1/4 or less than that of the nucleus, confirmed by focusing up and down and taking into account hepatocyte thickness. The authors found that repeated gavage with AlCl3 induced a significant increase in the numbers of micronucleated hepatocytes. Simultaneous administration of propolis attenuated the increased numbers MNHEPs induced by AlCl3. In addition, prolonged oral exposure to high doses of AlCl3 caused a significant increase in alkaline phosphatase, transaminases (AST and ALT) and lactate dehydrogenase (LDH) and induced histopathological changes in the liver. The authors suggested the observed clastogenicity after oral AlCl3 may have been mediated, at least in part, by free radicals (Abubakar et al., 2003). It must be noted here that there was no justification for the high oral dose examined and that the increased genetic damage occurred at doses that also induced overall systemic toxicity and cytotoxicity. As the report was written, it could not be excluded that genetic damage was associated with the oxidative stress in the liver as contrast to any direct clastogenic activity of Al. A Klimisch score of 2 was assigned to this study (weight of evidence study). The results of the study have limited utility in Al hazard identification.                     

     

Intraperitoneal injection

Geyikoglu et al. (2012) conducted a liver micronuclei (MN) assay to investigate genotoxic effects in adult male Sprague-Dawley rats (6 animals per group) treated daily with intraperitoneal injections of AlCl3at 5 mg/kg bw per day for 10 weeks. The control group of 6 rats received daily intraperitoneal injections of NaCl (the dose was not reported, but it can be assumed the solution was isotonic NaCl). The liver MN assay was performed in accordance with methods described by Turkez et al. (2010). Daily injections of AlCl3over 10 weeks resulted in a 4-fold increase in the numbers of micronucleated hepatocytes. The adverse effects on the liver and kidney and the histopathological changes in these organs suggested that the clastogenicity might be a consequence of the systemic toxicity and associated cytotoxicity. The reported findings supported previous observations on the genotoxic activity of AlCl3in mice following repeated intraperitoneal injections (Manna and Das, 1972). The Geyikoglu et al. (2012) study has following limitations: lack of detail on test material purity, test solution preparation and volume and pH of the administered solution. In addition there were no descriptions of clinical signs, drinking water or food consumption and body weight gain. No measures of Al levels in diet and drinking water were provided. A Klimisch score of 3 was assigned for this study (weight of evidence study).

Thus, on weight of evidence, aluminium compounds of normal particle size (i.e. not nanoparticles) do not induce genotoxic effects in somatic cells in vivo when administered by a physiologically relevant route.

 

Animal Studies – In-vivo Germ Cell Tests

Results from animal studies that have employed the dominant lethal assay are inconsistent. Guo et al.(2005) (Klimisch Score = 3) reported a positive response in rats subcutaneously dosed with AlCl3 daily (0, 7 and 13 mg Al/kg bw/day) for a two week period prior to 9 weeks of sequential matings.Two other studies (Dixon et al., 1979; Zelic et al., 1998), both of which were also assigned Klimisch Scores of 3, reported negative results, but the assessments of dominant lethal effects were very limited. Only Guo et al. (2005) reported aluminium levels in the testes indicating that aluminium had reached the target tissue. However, confidence in these results is limited by the high serum Al concentrations in the control group, the time delay in effect, the lack of information on the mating schedule and number of females, and the concurrent effects on fertility (fecundity, libido and male gamete histology).The evidence from animal studies for a mutagenic hazard to germ cells from aluminium ion is inconsistent and no clear conclusions about germ cell mutagenicity can be made based on these studies.

Since there are no known examples of substances inducing genotoxic effects in germ cells that are not genotoxic in somatic cells, it is highly unlikely that aluminium compounds pose any genotoxic risk to germ cells.

 

In-Vitro Gene Mutation Assays

The study by Oberly et al.(1982) (Klimisch Score = 2) was not considered sufficiently robust to meet this information requirement.Two recent gene-mutation studies conducted by Covance, Inc. (Covance, 2010b) using aluminium chloride (tested up to its solubility limit) and aluminium hydroxide (tested up to 10 mM in suspension and subsequently “cleaned” by Percoll density gradient centrifugation), did not find significant mutations at the thymidine kinase (tk) locus of mouse lymphoma L5187Y cells at any of the doses tested. This study type detects both gene mutations and chromosomal damage.These studies were conducted according to guidelines and GLP (Klimisch = 1 for aluminium chloride and 2 for aluminium hydroxide).

Sappino et al. (2011) investigated the potential genotoxicity of AlCl3in cultured MCF-10A cells and in cultured human primary mammary epithelial cells. Aluminium chloride hexahydrate (purity ≥ 99%) was dissolved in water at 1 M and immediately diluted to 10, 100 and 300 µM. The authors suggested that under these conditions, Al precipitation owing to polymerization was expected to be minimal and that no visual evidence of precipitates was observed. Stock solutions were diluted 1:1000 in fresh culture medium twice each week and an equivalent volume of water was used as the control. The addition of AlCl3had only a minor influence on the cell culture medium (control pH = 7.50±0.04; 10 µM: pH = 7.40±0.06; 100 µM: pH = 7.41±0.06; 300 µM: pH = 7.33±0.03). 

To investigate the role of Al in cell transformation, MCF-10A cells were cultured for 6 weeks in the presence of AlCl3  at 100 µM or in the presence of an equivalent volume of water. The authors suggest that “at the expected pH of the cell culture medium (pH ≈7.2), Al chloride and Al2Cl(OH)5 yield the same dissociation product, aluminum hydroxide”. Under these experimental conditions, AlCl3 induced a loss of contact inhibition and increased anchorage-independent growth of cultured MCF-10A cells. No effects of AlCl3on anchorage-independent growth were observed in HaCaT keratinocytes or on C26Ci human colonic fibroblasts cultured for 17 weeks in the presence of 300 µM AlCl3 compared to the same volume of water in controls. In a 7 day cell proliferation assay, AlCl3 at 100 or 300 µM reduced the numbers of MCF-10A cells at the density of 5000 cells per well in triplicate. Apoptosis measured using Annexin V staining revealed no differences between controls and MCF-10A cultures treated with up to 300 µM AlCl3 for 4 days. Exposure to 10, 100 or 300 µM AlCl3 increased the percentage of senescence-associated ß-galactosidase-positive cells in proliferating MCF-10A cultures after 7 days; at the same time, exposure to 100 or 300 µM AlCl3 increased the expression of p16/INK4a, a cyclin-dependent kinase inhibitor and tumor suppressor that enforces growth arrest and is known to increase with ageing in rodent and human tissues (Baker et al., 2011), in proliferating primary human mammary epithelial cells. The addition of AlCl3 increased DNA double strand breaks (DSBs) in a dose- and time-dependent manner in proliferating MCF-10A cells exposed to 10, 100 and 300 µM for 1 and 16 hours. Treatment with 100 and 300 µM AlCl3 increased DNA DSB in proliferating primary human mammary epithelial cells (p < 0.0001, two-sided t-test) but it had little or no effect on proliferating HaCaT keratinocytes (p = 0.21, two-sided t-test).Following X-ray irradiation there was no influence on the repair process of spontaneous DSBs in MCF-10A cells incubated for 16 hr in the presence of 300 µM AlCl3 compared to cells treated with the same volume of water. The cultured MCF-10A cells treated for 10 weeks with 10, 100 or 300 µM AlCl3 responded with upregulation of the p53/p21 pathway that mediates general and premature cell senescence. Based on the results of long-term culture of MCF-10A cells with high concentrations of AlCl3,Sappino et al.(2011) suggested that AlCl3 (up to 300 µM or 60 µM as Al) induced proliferation in MCF-10A cells, increased DSBs and accelerated senescence. According to the authors, the results indicated that induction of DSBs by AlCl3-treatment occurred slowly, suggesting that this effect was indirect and possibly cell-type specific.

There are a number of observations that can be made with regard to the Sappino et al. (2011) report. Other than comparisons to the Al concentrations found in commercial antiperspirants, there were neither justifications for the Al concentrations examined in cell cultures nor for the suggested correlations between Al2Cl(OH)5 and AlCl3 exposure. The Al concentrations examined in cell culture were far greater than the median 0.07 - 0.38 µM (< 10 µg/L) Al concentrations seen in the serum or plasma of healthy people (reviewed in Krewski et al., 2007) and do not reflect levels that could be achieved under normal circumstances.In the Sappino et al. (2011) study, no positive control groups were included, limited details were provided regarding the pH of the cell culture medium and it is not clear if the reported pH values were measured in the fresh or long-term culture medium. Weakly acidic conditions (pH 6.6-6.8) are generally mutagenic and clastogenic for cultured cells and these effects (even at non-cytotoxic concentrations) are the result of oxidative stress (e.g., increased free radicals and reactive oxygen species associated with lipid peroxidation). These changes often decline as cytotoxicity and cell death increase after exposure to higher levels of HCl and other acids (Morita et al., 1992). Sappino et al. (2011) pointed to positive results from other studies of Al genotoxic activity without consideration of the limitations of those studies in that many of the older studies used high concentrations of soluble Al compounds and the pH of the culture medium was not always controlled. Exposure of cultured cells to acidic media in many of the older studies could not be excluded (reviewed in Krewski et al., 2007) and abundant evidence exists to support the fact that acidic conditions in cultured human (Morita et al., 1992; Güngör et al., 2010) and rodent (Cifone et al., 1987; Morita et al., 1989; 1992) cells can increase the numbers of chromosomal aberrations (e.g., chromatid breaks and gaps). It has been established that AlCl3at neutral pH transforms to Al hydroxides including Al trihydroxide and Al oxidehydroxide and these hydroxides can precipitate from solution (Mayeux et al., 2012). The authors stated that they did not observe visually-evident precipitates (detection method was not reported); however, microscopic Al precipitates may have existed in the culture media (particularly at the highest concentration) and in that case, it might be that exposure of cells in culture to Al(OH)3 particulates occurred as compared to conditions in the control cultures.A Klimisch score 3 was assigned.

Turkez et al. (2011) conducted chromosome aberration (CAs) and sister chromatid exchange (SCEs) assays with alum (aluminum sulfate) in human lymphocytes. Experimental studies were conformed to the guidelines of the World Medical Assembly (Declaration of Helsinki). Whole heparinized blood samples were obtained from three healthy non-smoking donors with no history of exposure to any genotoxic agent. The Al2(SO4)3 concentrations tested were 0, 10 and 20 µg/mL (equivalent as 0, 1.57 and 3.15 µg Al/mL).The effects of bismuth subnitrate (BSN) on oxidative status of erythrocytes and cytogenetic changes in human lymphocytes were studied at 0.0625, 0.125, 0.25 and 0.5 µg/mL alone and applied to the cultures together with Al sulfate. Concentrations of Al2(SO4)3 at 10 µg/mL alone did not influence the frequency of SCEs and CAs; however, identical study with 20 µg/mL increased the frequency of SCEs per cell and CAs compared with controls. It must be noted here that no biologically significant increases (> 2-fold) of SCEs/cell were observed (publication Fig.1).Although there was no effect of Al2(SO4)3 at 10 µg/mL on oxidative stress markers in erythrocytes compared with the controls, the highest concentration of Al2(SO4)3 caused significant decrements in the activities of antioxidant enzymes (G-6-PDH, SOD and CAT) and reduced glutathione (GSH) in erythrocytes.. The authors suggested that increased SCEs and CAs resulted from the decreased activity of the antioxidant enzymes seen at 20 µg/mL. Concomitant treatment with BSN (except for 0.5 mg/mL) reduced the increased number of SCEs and CAs and the increased oxidative stress associated with Al2(SO4)3. Limitations to the Turkez et al. (2011) protocol include the fact only one time point was examined, reference mutagens were not included, only thirty well-spread metaphases were scored per sample for CA assay when OECD Test Guideline #473 requires that at least 200 well-spread metaphases be scored. In addition, the number of the second cycle metaphases examined for SCEs was not reported and the authors provided few details on laboratory methods. The highest Al concentration (3.15 µg/ml or 3,157 µg/L) examined was ~300x the Al concentrations (1.9-10.3 µg/L) present in normal human plasma and serum (Krewski et al., 2007). It should be noted that structural CAs can also occur as a result of cytotoxicity (Galloway et al., 2000) and in the presence of > 50% cytotoxicity, CA increases are most all artifactual and can represent false positives (Battersby et al., 2007 ; Kirkland et al., 2007; Galloway, 2000). The following criteria should be considered in selection of the highest concentration of the test substance: cytotoxicity, solubility of the compound in the test system, changes in pH and changes in osmolality (in OECD guideline for the in-vitro chromosome aberration test (OECD Test Guideline #473). Overall, the results of this study are equivocal. A Klimisch Score of 3 was assigned to this study.

 

In-Vitro Mammalian Cell Assays

The in-vitro micronucleus assay results of Migliore et al. (1999) for aluminium sulphate and the chromosome aberration assay of Lima at el. (2007) using aluminium chloride provide evidence that the aluminium ion is an in-vitro clastogen. Treatment of human lymphocytes with aluminum as AlCl3 has also been observed to induce oxidative DNA damage and inhibit repair of DNA damage from exposure to ionizing radiation (Lankoff et al., 2006). Caicedo et al. (2008), however, did not observe double DNA strand breaks at concentrations up to 5000 µM-Al (as AlCl3) in human jurkat T-cells, supporting an oxidative mechanism of action that produces single strand effects only. Available studies provide evidence for an indirect genotoxic mechanism of action for the aluminium ion involving the production of single strand breaks. An oxidative mechanism of action would be expected to exhibit a threshold, which may be expected to be higherin-vivodue to more efficient defence mechanisms than in cultured cells.

Thus, there is some evidence that soluble aluminium salts may induce DNA damage, probably by an oxidative mechanism, but these findings were not confirmed in recent GLP studies using the sensitive mouse lymphomatkassay. 

 

Other Relevant Information

In a weight of evidence assessment for a mutagenic effect in humans, the levels at which effects are seen in animal studies and the systemic bioavailability of the target substances need to be considered. The study conducted by Covance (2010b) in non-fasted rats observed no induction of micronuclei in bone marrow at the acute maximum tolerated dose (MTD) for aluminium hydroxide when administered by oral gavage, namely 2000 mg Al(OH)3/kg bw/day. The MTD had been determined previously in a range-finding experiment. Balasubramanyam et al. (2009a) also observed no statistically significant genotoxic effects in rat bone marrow after a single oral gavage of 2000 mg Al2O3/kg bw in the form of particles with a size-range of 50 to 200 µm. Although current toxicokinetic information does not allow the prediction of time profiles of levels of aluminium in target tissues as a function of realistic external exposures, when administered orally or by inhalation the target substances exhibit low bioavailability.

 

Conclusion for genetic toxicity

The available information does not provide indications for significant mutations at the thymidine kinase (tk) locus of mouse lymphoma L5187Y cells treated with aluminium hydroxide and aluminium chloride at any of the doses tested (Covance, 2010b; Oberly et al., 1982).

In vitro studies with human blood lymphocytes showed positive responses to aluminium sulphate for micronuclei formation (Migliore et al., 1999) and to aluminium chloride for the induction of chromosome aberrations (Lima et al., 2007). Aluminium chloride has also been shown to induce oxidative DNA damage in human lymphocytes (Lankoff et al., 2006). However, double DNA strand breaks were not observed at concentrations up to 5000 µM-Al (as aluminium chloride) in human jurkat T-cells (Caicedo et al., 2008). This supports an oxidative mechanism of action leading to single strand effects only. Thus, there is some evidence that soluble aluminium salts may induce DNA damage, probably by an oxidative mechanism.

The most relevant and methodologically strongest in vivo studies are those conducted by Covance (2010a) and by Balasubramnyam et al. (2009a, b).

In the Covance (2010a) study, the induction of micronuclei in the bone marrow was investigated in rats given aluminium hydroxide by oral gavage. No induction of micronuclei was observed up to the highest dose administered (2000 mg aluminium hydroxide/kg bw/day, corresponding to 700 mg Al/kg bw/day).

In the studies by Balasubramanyam et al. (2009a, b), the genotoxic effects of aluminium oxide particles were investigated in vivo. Single doses of aluminium oxide particulate suspensions were administered to rats by oral gavage. The study results were positive for the nano-sized materials with evidence of a dose-response relationship, while the genotoxicity levels for aluminium oxide bulk material (50 to 200 μm diameter particles) were not statistically significantly different from those for the control. The relevance of the results with nanomaterials for hazard assessment is unclear as the observed effects may have been related to the presence of nanoparticles as foreign bodies in the cells rather than to the chemical properties of the test material itself. Low toxicity, poorly soluble substances, such as aluminium oxide, have produced inflammatory effects in vitro, when present as nanoparticles. The proposed mechanism of action is the production of reactive oxygen species (ROS) (Donaldson and Stone, 2003; Nel et al., 2006; Oberdörster et al., 2005, 2007; Duffin et al., 2007; Dey et al., 2008). Current scientific knowledge does not allow differentiation of genotoxic effects due to the physical (nanoparticle) nature from genotoxic effects due to the chemical characteristics of the test substance (Landsiedel et al., 2009; Singh et al., 2009; Gonzalez et al., 2008). However, in the current scientific debate regarding the genotoxic effects of nanoparticles of many different substances, the possibility that nanoparticles stimulate an inflammatory response leading to oxidative stress in the cells and consequently to DNA damage is the most accepted hypothesis. Balasubramanyam et al. (2009a, b) reported tissue aluminium oxide levels elevated in a dose-response manner for the groups treated with nano-sized materials, consistent with transfer of the nano-sized particles across the gastrointestinal mucosa (Florence, 1997; Hagens et al., 2007). A particle size dependence of gastrointestinal absorption was apparent. Aluminium oxide levels in the tissues of animals dosed with the larger 50 to 200 μm diameter particles were not elevated to a statistically significant level, consistent with the notion of a low bioavailability of aluminium compounds (see Toxicokinetics).

Thus, on a weight of evidence approach, aluminium compounds in non-nanoparticle size ranges do not induce genotoxic effects in somatic cells in vivo when administered by a physiologically relevant route.

Taken together, the weight of evidence does not support a systemic mutagenic hazard for soluble and insoluble aluminium compounds. 

With regard to the nanosized material it is inconclusive whether the aluminium oxide shows a gentotoxic potential or not because the studies by Balasubramanyam et al. 2009a and b are not trustworthy due to the following reasons:

These two papers describe studies that have been well-designed and generally comply with the OECD guidelines that were in place at the time. Negative control CA, MN and comet data are normal, and positive control chemicals were effective. The results with the 30 and 40 nm samples of Al2O3 certainly suggest they are genotoxic, However, the data are so perfect they raise concerns. Such clear dose-responses for different endpoints over multiple sampling times are rarely seen following a single administration, and cannot be easily explained. The standard deviations, at least for the MN scores, are consistently to low to be credible taking normal inter-replicate and inter-animal variability into account. In order to confirm or refute the findings published by Balasubramanyam et al, it is recommended that a new combined MN and comet assay be performed, with oral dosing, to a robust OECD protocol. Therefore, these two in vivo studies are necessary for the correct assessment of aluminium oxide nano materials.

 

References not in IUCLID

Dey S, Bakthavatchalu V,et al.(2008). Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells. Carcinogenesis 29(10): 1920-1929.

Duffin R, Tran L, Brown D et al. (2007). Proinflammogenic effects of low-toxicity and metal nanoparticles In Vivo and In Vitro: Highlighting the role of particle surface area and surface reactivity. Inhalation Toxicology 19: 849-856.

Florence AT. (1997). The oral absoprtion of micro- and nanoparticulates: Neither exceptional or unusual. Pharmaceut Res 14(3): 259-266.

Gonzalez L, Lison D, Kirsch-Volders M. (2008).Genotoxicity of engineered nanomaterials: A critical review. Nanotoxicology 2(4): 252-273.

Hagens WI, Oomen AG, de Jong WH et al. (2007). What do we (need to) know about the kinetic properties of nanoparticles in the body? Reg Toxicol Pharmacol 49: 217 229.

Landsiedel R, Kapp MD, Schulz M, et al.(2009).Genotoxicity investigations on nanomaterials: Methods, preparation and characterization of test material, potential artifacts and limitations - Many questions, some answers. Mutat Res 681: 241-258.

Nel A, Xia T, Madler L, Li N (2006). Toxic potential of materials at the nanolevel, Science 311: 622-627

Oberdorster G, Oberdorster E, Oberdorster J (2005).Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles, Environmental Health Perspectives 113: 823-839

Oberdorster G, Stone V, Donaldson K (2007).Toxicology of nanoparticles: A historical perspective, Nanotoxicology 1: 2-25

Singh N, Manshian B, Jenkins GJS et al. (2009). Nanogenotoxicology: The DNA damaging potential of engineered nanomaterials. Biomaterials 3891-3914.

Justification for classification or non-classification

Based on the read-across from aluminium compounds within a weight of evidence approach (GLP-guideline studies) for genetic toxicity, no classification is required according to CLP (1272/2008/EC) classification criteria.