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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 bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
2020
Deviations:
yes
Remarks:
No test concentration (< 5 µg/plate) was insoluble in the final treatment mixture. No justificatin provided for not performing a confirmation of negative results; no data, if the efficacy of the S9 mix was chararcterized by a second mutagen.
GLP compliance:
not specified
Remarks:
The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: 97a
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- method of preparation of S9 mix: The S9 mix was prepared just before use by adding 900 µL of co-factors (5.2 mM D-glucose-6-phosphate, 4.4 mM nicotinamide adenine dinucleotide phosphate, 18 mM MgCl2, 28 mM KCl, 90 mM Na2HPO4.H2O, 8 mM NaH2PO4.H2O) and 100 µL S9 fraction.
Test concentrations with justification for top dose:
0.02, 0.04, 0.075, 0.15, 0.3, 0.6, 1.25 and 2.5 µg/plate with and without metabolic activation in all strains

The test material precipitated at concentrations higher than 2.5 µg/plate. Thus, this concentration was chosen as maximum one to test.
Vehicle / solvent:
- Vehicle used: DMSO/H2O 1:1

The test material was suspended in the vehicle and ultrasonicated for 10 min.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO/H2O 1:1 (v/v)
True negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: sextuplicates
- Number of independent experiments : single experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approx. 1E+09 bacterial cells/mL
- Test substance added in: preincubation mixture

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min at 37 °C
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies or a clearing of the background lawn in comparison with control plates

Evaluation criteria:
A positive response in the test was defined as an increase (at least twofold above the control) in His+/wild revertant colonies in every strain.
Statistics:
Mean values and standard deviation were calculated. The one-way analysis of variance (one-way ANOVA), followed by Dunnett’s multiple comparison post test, was used to verify the significance of a positive response. A P value < 0.05 was considered statistically significant.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
STUDY RESULTS
- Concurrent positive control data : The positive control substances (SA, 2NF, 9AA, mitomycin C and 2AA) at µg levels increased the number of revertant colonies by several fold compared to the control, revealing the sensitivity of the system to detect a mutagenic effect.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : Even at the highest tested concentration no increase in the number of revertants was detected and thus no concentration-response relationship is evident.
- Statistical analysis; P-value : All positive control substances resulted in P < 0.01 vs. control (ANOVA).


Ames test:
- Signs of toxicity : All concentrations tested did not show any statistically significant decrease in the number of revertant colonies compared to the control and thus, they resulted in lack of cytotoxicity.
- Individual plate counts : no data
- Mean number of revertant colonies per plate and standard deviation : please refer to table 1


HISTORICAL CONTROL DATA: no data

Table 1. Test results for Al2O3-bulk

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate (n=6)

± standard deviation

TA100

TA1535

TA98

TA97a

TA102

+

DMSO (50 µL/plate)

153.3 ± 14.9

12.6 ± 5.6

23.3 ± 4.5

178.3 ± 11.5

320.6 ± 21.5

+

DMSO/H2O 1:1 (v/v)

146.4 ± 16.2

13.3 ± 4.9

26.1 ± 5.3

169.2 ± 10.2

311.2 ± 22.2

+

20

154.6 ± 10.9

12.4 ± 3.2

24.3 ± 4.5

177.1 ± 14.5

319.0 ± 23.1

+

40

151.3 ± 9.0

13.4 ± 3.8

26.5 ± 4.0

178.6 ± 14.2

320.8 ± 21.3

+

75

154.7 ± 10.5

11.9 ± 5.8

24.6 ± 4.1

179.6 ± 11.8

322.1 ± 19.3

+

150

157.3 ± 10.1

12.6 ± 3.7

26.7 ± 4.8

176.2 ± 15.1

323.6 ± 21.1

+

300

152.2 ± 9.7

12.3 ± 6.1

24.1 ± 3.6

175.3 ± 11.5

320.3 ± 39.1

+

600

161.3 ± 10.5

13.3 ± 4.9

26.9 ± 6.2

175.3 ± 16.4

320.6 ± 27.2

+

1250

166.3 ± 11.2

13.9 ± 5.1

25.3 ± 5.5

171.2 ± 16.1

327.3 ± 24.5

+

2500

169.7 ± 9.8

13.2 ± 4.4

26.6 ± 4.4

174.5 ± 14.8

323.4 ± 22.3

+

Positive controls

2AA

2AA

2AA

2AA

2AA

Mean No. of colonies/plate

± SD

695.6 ± 32.9*

432.4 ± 27.9*

208.9 ± 17.2*

830.6 ± 36.4*

1212 ± 82.5*

-

DMSO (50 µL/plate)

102.3 ± 17.5

16.6 ± 5.0

25.3 ± 3.1

174.0 ± 19.8

254.0 ± 25.0

-

DMSO/H2O 1:1 (v/v)

104.6 ± 10.2

14.4 ± 3.9

23.1 ± 3.3

169.6 ± 15.5

241.2 ± 22.2

-

20

106.0 ± 14.3

18.0 ± 3.0

26.1 ± 3.1

171.6 ± 14.

241.1 ± 20.2

-

40

99.3 ± 14.2

17.2 ± 5.2

23.3 ± 2.5

184.0 ± 24.5

264.0 ± 21.6

-

75

112.6 ± 8.5

15.0 ± 5.1

26.3 ± 3.5

171.3 ± 19.6

251.0 ± 25.7

-

150

108.3 ± 10.5

16.0 ± 2.6

23.3 ± 3.1

180.0 ± 22.0

267.3 ± 31.7

-

300

116.0 ± 11.3

16.6 ± 4.1

25.1 ± 3.7

172.3 ± 19.0

268.6 ± 28.8

-

600

126.0 ± 13.7

18.3 ± 3.5

24.3 ± 3.5

177.3 ± 17.7

277.3 ± 29.1

-

1250

114.4 ± 12.3

17.7 ± 2.9

25.2 ± 2.8

180.1 ± 16.8

268.6 ± 30.1

-

2500

121.4 ± 14.2

19.1 ± 3.1

26.7 ± 3.3

182.1 ± 15.4

271.4 ± 33.1

-

Positive controls

SA

SA

2NF

9AA

Mitomycin C

Mean No. of colonies/plate

± SD

483.6 ± 53.5*

323.3 ± 26.5*

95.3 ± 8.6*

625.6 ± 43.4*

1101 ± 84.02*

SA = sodium azide

9AA = 9-aminoacridine

2NF = 2-nitrofluorene

2AA = 2-aminoanthracene

* P < 0.01 vs. Control (ANOVA)

Conclusions:
Al2O3-bulk was negative in the Ames test in the S. typhimurium strains TA 97a, TA 98, TA 100, TA 1535 and TA102 up to and including a concentration of 2500 µg/plate. No cytotoxicity nor precipitation was noted in any of the tested concentrations.
Endpoint:
genetic toxicity in vitro, other
Remarks:
cytogenicity (chromosome aberration and micronucleus study)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
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
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
Remarks on result:
other: woe, source, RA-A, 7446-70-0, Lima et al., 2007
Remarks:
chromosome aberration
Conclusions:
The source substances (CAS 7446-70-0 and 10043-01-3) were positive in the micronucleus assay as well as in the chromosome aberration assay without metabolic activation. Applying the read-across approach, similar results are expected for the target substance (CAS 1344-28-1).
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
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
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
Remarks on result:
other: woe, source, RA-A, 7446-70-0, Oberly et al., 1982
Conclusions:
The source substances (CAS 7446-70-0 and 21645-51-2) did not exhibit mutagenic properties in mammalian cells. Applying the read-across approach, the target substance (CAS 1344-28-1) is expected to have a similar mutation potential in mammalian cells.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
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
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
Remarks on result:
other: woe, source, RA-A, 7784-13-6, Caicedo et al., 2008
Conclusions:
The source substance (CAS 7784-13-6) was negative in the comet assay without metabolic activation in one study but positive in the comet assay without metabolic activation in another study. Applying the read-across approach, similar results are expected for the target substance (CAS 1344-28-1).

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 - 100 g
- Housing: Rats were kept in polypropylene cages under controlled conditions
- Diet: Animals received a 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%), ad libitum.
- Water: Animals received a standard laboratory water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 60 ± 10
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12
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: 30 h, 48 h

Mitotic Index (MI): 18 h, 24 h

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 - 0.3 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:
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
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.
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 18 h:
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 24 h:
Negative control: 3.25 ± 0.10
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

30 h: 2.5 ± 0.70

48 h: 1.8 ± 0.75

Al2O3- (50 - 200 μm)

30 h 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 30 h and 48 h were similar.

30 h:

25 mg Al: 1.9±0.73, ns

50 mg Al: 3.3±1.16, ns

100 mg Al: 5.9±1.71, ns

48 h:

25 mg Al: 2.0±0.64, ns

50 mg Al: 4.2±1.07, ns

100 mg Al: 6.6±1.68, ns

Conclusions:
The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those of the vehicle control.

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) 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. The results were negative for the 50 to 200 µm sized Al2O3-bulk particles.

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. 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 in levels of Al in tissues and organs with increasing dose. 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 / 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
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 - 100 g
- Housing: Rats were kept in polypropylene cages under controlled conditions
- Diet: Animals received a 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%), ad libitum.
- Water: Animals received a standard laboratory water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 60 ± 10
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12
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: 18 h, 24 h

Mitotic Index (MI): 18 h, 24 h

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
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.
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 18 h:
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 24 h:
Negative control: 3.25 ± 0.10
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)

18 h: 0.6 ± 0.32

24 h: 0.5 ± 0.42

 

Al2O3- (50 - 200 μm)

18 h:

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)

24 h:

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)

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

Conclusions:
The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those of the vehicle control.

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 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. 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. 

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.

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. 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. 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
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 - 120 g
- Housing: Rats were kept in polypropylene cages under controlled conditions
- Diet: Animals received a 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%), ad libitum.
- Water: Animals received a standard laboratory water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 60 ± 10
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12
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: 48 h, 72 h

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
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

48 h: 1.51 ± 0.93

72 h: 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.

48 h:

25 mg Al: 1.75 ± 0.67, ns

50 mg Al: 1.98 ± 0.98, ns

100 mg Al: 3.99 ± 1.29, ns

72 h:

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.

Conclusions:
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.

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. 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. No statistically significant effect was evident for Al2O3-bulk.

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. 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:
in vivo mammalian cell study: DNA damage and/or repair
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 for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
The alkaline Comet Assay is a sensitive assay that detects DNA damage at the level of a single cell. DNA damage such as single strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking and single strand breaks from impaired excision repair can be detected (Tice et al., 2000). Cells are lysed to liberate DNA, exposed to alkali, undergo electrophoresis, and then neutralization. The DNA is then stained, viewed and the “comet” shapes scored according to the size of the tail and/or % of DNA in the tail as an index of overall DNA damage. Cytotoxicity must be measured concurrently.
GLP compliance:
not specified
Remarks:
The investigation was reported in a scientific paper without specifying whether GLP conditions were applied.
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 90 - 120 g
- Housing: Rats were kept in polypropylene cages under controlled conditions
- Diet: Animals received a 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%), ad libitum.
- Water: Animals received a standard laboratory water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 60 ± 10
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12
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:
Comet Assay: 4 h, 24 h, 48 h, 72 h
(Tice et al. 2000: 3 - 6 h and 22-26 h)

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 amount of 47.5 mg Al2O3 assuming a body weight of ca. 100 g equivalent to 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 ca. 100 g equivalent to 50 mg Al
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Al2O3; corresponding to an actual amount of 190 mg Al2O3 assuming a body weight of ca. 100 g equivalent 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:
Comet Assay:
Three slides were prepared per sample. Singh et al. (1988) and Tice et al. (2000) were cited in the paper for these methods. Ethidium bromide was used as a stain and 150 cells per rat (50 cells per slide) were scored using a fluorescence microscope. Quantification of DNA damage was reported as the % Tail DNA and was assessed using Comet Image Analysis System (version 5.5, Kinetic Imaging Ltd. Nottingham, UK).

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: 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).
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
Toxicity:
no effects
Remarks:
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.
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).

General toxicity: no detail provided

Comet Assay:

% Tail DNA

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

4 h: 0.6 ± 0.32

24 h: 0.5 ± 0.42

Al2O3- (50 - 200 μm)

4 h:

25 mg Al: 2.42 ± 1.14 - ns

50 mg Al: 3.80 ± 2.61 - ns

100 mg Al: 5.40 ± 3.59 – ns

24 h:

25 mg Al: 4.68 ± 2.44 - ns

50 mg Al: 5.26 ± 2.48 - ns

100 mg Al: 7.26 ± 4.53 - ns

Conclusions:
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.
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) used the alkaline Comet Assay (percent comet tail DNA estimated from 150 cells per rat) 4, 24, 48 and 72 hours after a single dose of 50 to 200 µm Al2O3-bulk material. Cell viability, assessed using the trypan blue exclusion assay and reported only qualitatively in the article, was > 80% at all time points. Consistent with the results of other genotoxicity / mutagenicity assays conducted by these authors, no dose-response relationship was evident for the animals trated with the bulk material.

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. 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:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Documentation is insufficient for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Version / remarks:
no guideline was available at the time of the study but the design was clearly stated as a dominant lethality study.
Deviations:
yes
Remarks:
: only 1 dose tested; lack of details on test substance; number of pregnant females per dose group was not provided
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
Nude Balb/cAnN
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: Females (n=720) were 8-10 weeks old and the males (n=45) were 10-12 weeks old
- Weight at study initiation: no data
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12h light/dark cycle

Additional information
Method of allocation to groups:
Prior to the main experiment, the fertility of a larger group of male mice was determined by a serial mating experiment. Two virgin females were caged with each male mouse for 7 days. After 7 days, the female mice were replaced with two more virgin females. This procedure was repeated for 4 pairs of female mice. Female mice were killed by cervical dislocation at gestation day 13 and the uteri examined for live and dead foetuses.

The male mice identified as fertile by this process were then randomly allocated to one of three experimental groups.

Further details on study design:
Mating schedule: Two female mice were housed with a fertile treated male for 7 days before replacement with another two females. This continued for 8 cycles to give a total duration of 8 weeks, sufficient for spermatogenesis to occur.

Females were killed on gestation day 13.
Route of administration:
intraperitoneal
Vehicle:
phosphate buffered saline; the pH was not reported
Details on exposure:
no further information
Duration of treatment / exposure:
Exposure Duration: Single
Frequency of treatment:
Schedule/Frequency of Administrations:
24 hours prior to caging with females.
Post exposure period:
DLT Study Outcomes:
Weekly, weeks 1 to 8

General toxicity: not reported
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
DLT Study
Males:
15 fertile mice per dose group

Number of females: 720 in total. The number of pregnant mice per mating interval was not reported.
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl-methyl-sulphonate (Endoxane, Asta Medica, Germany)
Justification: not provided
Route of administrations: i.p.
Dose/concentration: 200 mg/kg bw
Tissues and cell types examined:
Live-dead assay fetuses and fetal malformations
Details of tissue and slide preparation:
Details of fixing and examination were not provided. Examination was blinded.
Evaluation criteria:
Zelic et al. (1998) reported their results as the “reproductive potential” i.e., apparently the number of live embryos per female animal. The criterion for a positive response was not stated explicitly but the results were clearly negative.
Statistics:
2v2 contingency tables and χ2 were used to compare treated versus control; ANOVA was used to assess inter-group differences when three variables were considered. Adjustment of p-values for multiple comparisons does not appear to have been considered.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Dead foetuses per female: not reported

 

Live foetuses per female (mean number of embryos±sem):

Negative Control (buffer + Tween80)

Week 1: 4.77 ± 1.06

Week 2: 4.93 ± 1.10

Week 3: 4.33 ± 0.96

Week 4: 4.70 ± 0.99

Week 5: 4.37 ± 1.06

Week 6: 3.13 ± 1.10

Week 7: 3.97 ± 1.01

Week 8: 3.89 ± 0.96

 

Treated Group (Aldovit)

Week 1: 7.17 ± 0.73

Week 2: 5.37 ± 1.04

Week 3: 4.93 ± 0.91

Week 4: 3.83 ± 0.95

Week 5: 5.67 ± 0.98

Week 6: 3.80 ± 0.93

Week 7: 2.37 ± 0.91

Week 8: 3.20 ± 1.01

 

Positive Control ()

Week 1: 2.70 ± 0.68

Week 2: 2.57 ± 0.84

Week 3: 2.40 ± 0.85

Week 4: 3.43 ± 0.89

Week 5: 3.23 ± 0.91

Week 6: 1.47 ± 0.72

Week 7: 1.93 ± 0.78

Week 8: 2.20 ± 0.87

Conclusions:
Mice treated with Aldovit exhibited similar numbers of viable embryos as the animals treated with the negative control (Tween 80).
During week 1 post-exposure, the reproductive potential of the animals treated with Aldovit was higher than the negative control. A decrease in reproductive potential with time was evident. Animals treated with endoxane, the positive control, showed lower numbers of embryos during the first 3 weeks post-exposure. The treatment group curves converged with increasing number of mating cycles and the variability between animals increased. The results for Aldovit were negative but the limitations of the study and the variability in the results limit confidence in the results.
Executive summary:

Zelic et al. (1998) treated fertile male BALB-cAn NCR mice (10-12 weeks old) with a single intraperitoneal injection of a suspension of aluminium oxide (Aldovit, a ceramic dental implant material) (1 g/kg bw), ethyl-methane-sulphonate (EMS) at a concentration of 200 mg/kg (positive control), or Tween-80 at a concentration of 10 mg/kg (a nonionic surfactant and emulsifier of polysorbate, negative control) 24 hours prior to housing with untreated, virgin female mice. The vehicle was phosphate buffered saline. Two female mice were housed with a fertile treated male for 7 days before replacement with another two females. This continued for 8 cycles to give a total duration of 8 weeks, sufficient for spermatogenesis to occur. Pregnant female mice were killed on day 13 of gestation. Zelic et al. (1998) reported their results as the “reproductive potential” i.e., apparently the number of live embryos per female animal. In females fertilized during the first week post-injection, male partners treated with Aldovit exhibited the highest number of viable embryos. For the remainder of the sequential mating cycles, there were no significant differences in the mean number of viable embryos in females impregnated by mice treated with Tween-80 (negative control) and Aldovit. Mice treated with the positive controlexhibited lower reproductive potential than both Aldovit and Tween-80 during the first three weeks post-exposure. The results of this study with respect to the DLT were not reported in the recommended manner; details are insufficient. Only one dose level of the Aldovit was administered.

The variability of measurements was high. It is also unclear what dose of aluminium reached the testes. A Klimisch Score of 3 has been assigned.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: woe, source, RA-A 21645-51-2, Covance Laboratories Ltd, 2010a
Conclusions:
The source substance (CAS 21645-51-2) was negative in the micronucleus assay. Applying the read-across approach, similar results are expected for the target substance (CAS 1344-28-1).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro there is only one study with aluminium oxide (bulk form) available (Ames test), 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 the 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 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

Aluminium oxide in the bulk- and nanoform (Al2O3-30 nm and Al2O3-40 nm) was assessed in a bacterial reverse mutation assay (Ames test) according to OECD Guideline 471 (Balasubramanyam et al., 2010). The mutagenic potential of the test substances was examined in S. typhimurium tester strains TA 97a, TA 98, TA 100, TA 1535 and TA 102 at concentrations up to 2500 µg/plate in one experiment (pre-incubation method) with 6 replicates. In a preliminary experiment precipitation of the test substances in DMSO/H2O 1:1 (v/v) occurred at concentrations higher than 2500 µg/plate. The test substances did not induce an increase in reversions in any of the tested strains with or without metabolic activation. Cytotoxicity was not observed. The vehicle and positive controls proved the validity of the experiment. Thus the bulk form as well as the two nanoforms of aluminium oxide (Al2O3-30 nm and Al2O3-40 nm) were not considered mutagenic in bacteria.

 

Further 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). 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, these available mutagenicity assays have tended to use aluminium (III) salts that are more soluble than the target substance. 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.

 

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 particle 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 genotoxicity levels for 50 to 200μm diameter particles (Al2O3-bulk) were 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 exposure 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.I 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 AlCl3 at 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 AlCl3 over 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 support previous observations on the genotoxic activity of AlCl3 in mice following repeated intraperitoneal injections (Manna and Das, 1972). The Geyikoglu et al. (2012) study has the 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 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 score 1 for aluminium chloride and 2 for aluminium hydroxide).

Sappino et al. (2011) investigated the potential genotoxicity of AlCl3 in 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 AlCl3 had 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, aluminium 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 AlCl3 on 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 AlCl3 at 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 (CA) and sister chromatid exchange (SCE) assays with alum (aluminium 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 to 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 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 while OECD 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 likely artifacts 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 the OECD guideline for the in-vitro chromosome aberration test (OECD 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 aluminium 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 higher in-vivo due 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 lymphoma tk assay.

 

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 Balasubramanyam 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 particle 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 200μm 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 200μm diameter particles were not elevated to a statistically significant level, consistent with the notion of a low bioavailability of aluminium compounds (see Toxicokinetics).

Thus, based on a weight of evidence approach, it is concluded that 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. 

 

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

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.