Aluminium hydroxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

In vivo genotoxicity assessment of aluminium oxide nanomaterials in rat peripheral blood cells using the Comet Assay and micronucleus test

Author:

Balasubramanyam A, Sailaja N, Mahboob M, Rahman MF, Misra S, Hussain SM, Grover P.

Year:

2009

Bibliographic source:

Mutagenesis 24(3): 245-251.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Al2O3 (bulk)

IUPAC Name:

Al2O3 (bulk)

Constituent 2

Reference substance name:

Aluminium oxide

EC Number:

215-691-6

EC Name:

Aluminium oxide

Cas Number:

1344-28-1

Molecular formula:

Al2O3

IUPAC Name:

aluminium oxide

Details on test material:

- Name of test material (as cited in study report), source and purity: Al2O3 (bulk): Sigma-Aldrich, 70 - 290 mesh (50 - 200 μm); purity >90%Al2O3 (30 nm): NovaCentrix, product M1056; purity >90%Al2O3 (40 nm): NovaCentrix, product M1049-D; purity >90%Al2O3 (bulk): not described furtherAl2O3 (30 nm): mean±sd - 39.85 ± 31.33 nmAl2O3 (40 nm): mean±sd – 47.33 ± 36.13 nmParticle size measurement method: TEM and dynamic light scattering (DLS) using NMs suspended in 1% Tween 80 in MilliQ water. Fresh suspensions were used with ultrasonification for 10 minutes prior.Particle charge: Laser Doppler Velocimetry (LDV)Particle shape: roughly sphericalLevels of specific contaminants: Not reported.Phosphate buffered saline was mentioned in the description of chemicals used but was not referred to further in the article.

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system 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 weekENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 3ºC- Humidity (%): 60 ± 10%- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles

Administration / exposure

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, 72hCell viability (trypan blue exclusion assay): not reported further Al Level Study:Urine and faeces: 48 h after dosingBlood and tissues: 14 days after dosing

Doses / concentrations

Remarks:

Doses / Concentrations:0, 500, 1000 and 2000 mg Al2O3/kg bw (actual amounts of 47.5 mg, 95 mg and 190 mg Al2O3 assuming a body weight of ca. 100 g equivalent to 25, 50 and 100 mg Al)Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide monohydrateRoute of administration(s): intraperitoneal Dose/concentration: 40 mg/kg bw, volume= 0.01 mL/g bw

Examinations

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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).

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive 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.