Aluminium triisopropanolate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Upon contact with water or moisture (e.g. within mucous membranes) aluminium tri-isoproanolate hydrolyses immediately to isopropanol and aluminium 3+ cations (as hydroxide and oxyhydroxide). Hence, toxicity is determined by the toxicity of these two species.

In a test according to OECD 471 Salmonella typhimurium strains TA 100, TA1535, TA98, TA97a and TA102 were exposed to aluminium oxide with and without metabolic activations. No increase in the number of revertants was observed in any of the strains tested. Therefore it is concluded that the substance is not mutagenic (Balasubramanyam 2010).

No forward mutations at the thymidine kinase (tk) locus in the L5178Y mouse lymphoma assay with the use of AlCl3 at concentrations from 2.36-2.59 mM were observed. The lowest relative survival observed in the study was 38% at 600 µg/mL, which is considered insufficient to allow a valid definitive conclusion (Oberly 1982).

DNA damage in human (Jurkat) T-cells at concentrations of 50, 100, 500, 1000 and 5000 µM-Al (AlCl3) using the neutral Comet Assay. Aluminium did not result in significant increases in DNA double strand breaks. The pH used for lysing and unwinding was not clear from the report in the article (Caicedo 2008).

In an Ames test isopropanol was non-mutagenic in TA100, TA1535, TA1537, TA97 and TA98 with and without metabolic activation (S9) (Zeiger 1992

In an in vitro HGPRT test with and without metabolic activation isopropanol did not induce clastogenic effects (Young 1990)

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

data published

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

purity > 90%
particle size: 50-200 um

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 98, TA 97a, TA102

Metabolic activation:

with and without

Metabolic activation system:

S9 from male rat liver (no details)

Test concentrations with justification for top dose:

20, 40, 75, 150, 300, 600, 1250 and 2500 ug/plate
Based on the presence of precipitate above 2500 ug/plate

Vehicle / solvent:

vehicle:DMSO/ H2O (1/1)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO/H2O

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: aminoanthacene

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation
- Cell density at seeding (if applicable): 1E09 cells/mL

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 6

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn, reduction of revertant colonies

Rationale for test conditions:

standard according to OECD 471

Evaluation criteria:

significant increase of number of revertant colonies compared to controls

Statistics:

NA

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA97a, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The substance is not mutagenic in the Ames test

Executive summary:

In a test according to OECD 471 Salmonella typhimurium strains TA 100, TA1535, TA98, TA97a and TA102 were exposed to aluminium oxide with and without metabolic activations. No increase in the number of revertants was observed in any of the strains tested. Therefore it is concluded that the substance is not mutagenic.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Comet assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

no guideline available

Principles of method if other than guideline:

The Comet Assay depends on the ability of negatively-charged fragments of DNA to undergo electrophoresis in an agarose gel. The extent to which DNA migrates correlates directly with the amount of DNA damage. A suspension of cells is mixed with low melting point agarose, spread on a slide and lysed. DNA is then unwound and undergoes electrophoresis at a particular pH. The pH used dictates the types of DNA damage detected. For example, a neutral pH (7-8), as used in this study, detects predominantly double strand breaks and cross links while a pH>13 allows detection of single strand breaks, incomplete excision repair sites, and alkali labile sites (ALS) in addition to the lesions detected at neutral pH. Under the influence of an electric field, the damaged DNA migrates towards the anode producing a shape like a comet. The amount of migration and shape of the comet are an index of the DNA damage that can then be analysed microscopically or with image analysis software.

GLP compliance:

not specified

Type of assay:

comet assay

Target gene:

Not applicable.

Species / strain / cell type:

other: CD4+T cells

Details on mammalian cell type (if applicable):

Species/Cell Line/Source of Cells:
Human CD4+ T cells were obtained from a T-helper lymphoma Jurkat cell line from American Type Culture Collection (Manassas, VA, USA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 50, 100, 500, 1000 and 5000 μM AlCl3.

Vehicle / solvent:

No information.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

other: different salts tested

Positive control substance:

no

Details on test system and experimental conditions:

Cell culture processing & conditions:
Cells were cultures in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) at 37 ºC in a atmosphere composed of 0.5% CO2. After they became confluent (every 3 to 4 days), the cultures were subcultured. Cells were washed twice with PBS and resuspended in 24 well-plates containing fresh medium and the test solutions.

Preparation of test solutions:
No information.

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

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

Analytical verification of dose levels:
Not carried out.

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

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

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

Evaluation criteria:

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

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

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

Proliferation inhibition [3H-thymidine uptake]:
P<0.05 comparing metal-treated cells counts per minute with the untreated control.

Statistics:

Student’s t-test and a significance level of p<0.05.

Species / strain:

other: CD4+T cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Remarks:

other salts

Additional information on results:

- cytotoxicity
Cell viability was determined using propidium iodide (PI) staining and in a FACScan flow cytometer (Becton Dickinson Co.) General viability/toxicity for the different metal treatments were ranked using an LC50 index (half lethal concentration, i.e., the concentration at which 50% of the cells were viable). These tests were conducted in triplicate.

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

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

Primary Endpoint(s):

Neutral Comet Assay

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

Apoptosis (flow cytometry/caspase-9)

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

 

Significant detriment to viability or ‘toxicity”:

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

 

Proliferation inhibition [3H-thymidine uptake]:

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

Other salts:

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

Metal	DNA damage	Apoptosis (Caspase-9)	Viability (PI)	Prolife-ration Inhibition
V	50	50	1000	50
Ni	50	100	5000	500
Co	5000	5000	500	100
Cu	>5000	500	5000	100
Nb	>5000	500	500	>5000
Mo	>5000	1000	>5000	500
Zr	5000	500	5000	>5000
Be	>5000	5000	1000	5000
Cr	>5000	>5000	>5000	>5000
Al	>5000	5000	>5000	>5000
Fe	>5000	5000	>5000	>5000

Conclusions:

Aluminium treatment did not result in significant increases in DNA double strand breaks (neutral Comet Assay)
In addition no inhibition of cell growth (3H-thymidine uptake), effects on viability (PI dye that stains nuclear DNA) was observed at any concentration applied. A significant apoptotic effect determined through measurement of intracellular caspase-9 activity was observed only at the highest concentration of AlCl3 used (5000 µM-Al).

Significant genotoxic effects were observed for vanadium, nickel and cobalt. Based on DNA damage, apoptosis, and effects on viability and prolfieration, the authors ranked the metal chloride salts as follows for harmful effects: V>Ni>Co>Cu>Nb>Mo>Zr>Be>Cr>Al>Fe.

Executive summary:

Caicedo et al. (2008) examined DNA damage in human (Jurkat) T-cells at a range of concentrations of AlCl3 (50, 100, 500, 1000 and 5000 µM-Al) using the neutral Comet Assay. Unwinding and electrophoresis of DNA at neutral pH detects fewer types of DNA damage compared with use of pH > 13. The neutral assay detects predominantly double strand breaks. A significant effect for DNA damage was defined as an index of DNA damage (based on relative comet tail length) of >75. Aluminium did not result in significant increases in DNA double strand breaks. Overall the study appeared to follow a standard protocol and the results were negative.

In the publication, cell viability was measured using propidium iodide staining, cell proliferation using 3H-thymidine uptake and apoptosis using caspase-9 immunostaining and flow cytometry.

More than 50% caspase-positive cells was considered significant apoptosis and >50% propidium iodide positive cells as a significant effect on viability or a significant effect on viability at any concentration applied. Apoptosis was significant only at 5000 µM-Al. The cells used in this study have not been extensively used with this assay. If the p-value from the Student’s t-test was less than 0.05 in the comparison of the counts per minute in metal-treated cells compared to untreated controls, the inhibition of proliferation based on 3H-thymidine uptake was considered significant. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

: lack of details on test substance

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Line/Source of Cells:
TK+/- -3.7.2 heterozygote of L5178Y mouse lymphoma cells.

Medium:
Fischer’s medium for leukemic cells mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G, streptomycin sulfate.

Metabolic activation:

without

Metabolic activation system:

S9-mix (from livers of aroclor treated rats)

Test concentrations with justification for top dose:

570, 580, 590, 600 and 625 µg/mL

Vehicle / solvent:

Sterile, deionized, glass-distilled water

Untreated negative controls:

other: Sterile, deionized, glass-distilled water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: EMS (ethyl methanesulphonate) and AAF ( N-2-fluorenylacetamide (2-acetylaminofluorene))

Details on test system and experimental conditions:

Cell culture processing & conditions:
- cells were thawed from frozen stock, maintained in Fischer’s medium for leukemic cells from mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G, streptomycin sulphate.
- background spontaneous TK-/- mutant frequencies were reduced by weekly 24 hours treatment with medium containing thymidine, hypoxanthine, methotrexate and glycine.

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

Administration of test solutions:
Volume: 0.1 mL

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

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

Incubations per dose/time point:
2 or more

Metabolic activation:
10% dilution of S9 in medium; added with cofactor mix containing NADP at 8 mg/mL and isocitric acid (at 15 mg/mL). A test with S9 was only reported when showing a positive response

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

Ancillary endpoints examined:
Cell viability was determined by trypan blue dye exclusion.
Total survival: by the method of Clive and Spencer (1975) – combining growth in suspension culture and soft cloning efficiency data.

Evaluation criteria:

Mutant frequency (MF): number of mutants per 105 colony forming cells.

Positive response definition:
“a test agent will be considered positive in the L5178Y mouse lymphoma assay if a dose-related response is obtained in which two or more concentrations elicit a greater then 2-fold increase in MF over the solvent with a minimum of 10% survival (Clive et al., 1979).”

Statistics:

No data.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was a large change in pH when AlCl3 was added to the medium with possible effects on growth.


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


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

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

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

 

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

Conclusions:

Based on the conditions of the test AlCl3 is considered not mutagenic to mammalian cells.

Executive summary:

In this test no forward mutations at the thymidine kinase (tk) locus in the L5178Y mouse lymphoma assay with the use of AlCl3 at concentrations from 2.36-2.59 mM were observed. The study was well-described and used a standard assay but did not test a suitable range of concentrations. At least 4 analysable concentrations should be used with the maximum resulting in 10-20% relative survival (OECD TG#476, 1997). The lowest relative survival observed in the study was 38% at 600 µg/mL. A further caveat on the reliability of the results for AlCl3 is the observation of a large pH change on addition of this substance to the medium, a change that may influence growth. 

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, according to accepted guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver microsomes (S9)

Test concentrations with justification for top dose:

Range-finding (cytotoxicity) assay: 0.0098, 0.0195, 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.5, or 5.0 mg/mL
Mutation assay (- & + S9): 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 mg/mL (Trial 1)
Mutation assay (- S9): 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, or 5.0 mg/mL (Trial 2)
Mutation assay (+ S9): 0.5, 1.0, 2.0, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/mL (Trial 2)
Mutation assay (- S9): 1.0, 2.0, 3.0, 4.0, 4.5, or 5.0 mg/mL (Trial 3)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile, deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile, deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 5-bromo-2-deoxyuridine (-S9) and 3-methylcholanthrene (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours at 37 ºC
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 to 10 days at 37 ºC

NUMBER OF REPLICATIONS: 3 (-S9) or 2 (+S9) independent experiments.

Evaluation criteria:

(a) A dose- or toxicity-related increase in mutant frequency must be observed for at least 3 doses.
(b) A mutagenic dose-response in one assay should be confirmed in a second mutation assay.
(c) If an increase in mutant frequency is observed in one trial for a treated culture near the highest testable toxicity and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test article generally will be considered to be mutagenic.
(d) Applied concentration or toxicity (% survival) can be used to establish whether the mutagenic activity is related to an increase in effective treatment.
(e) Treatments that reduce relative clonal survival to less than 5% may be included in the assay but will not be used as sufficient evidence of mutagenicity as it relates to risk assessment.

Statistics:

Not performed (colonies are counted)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

negative with metabolic activation
negative without metabolic activation

The test material, isopropanol, is considered negative for inducing forward mutations at the HGPRT locus in CHO cells both in the presence and absence of S9 metabolic activation as shown in this test.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation. Tn.publication is sufficiently detailed to allow a scientific evaluation of the results

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

- S. typhimurium strain TA102 or E. coli strain WP2 uvrA were not used & 2-aminoanthracene was the only positive control compound used to test the efficacy of the S9 fraction.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster liver microsomes (10% and 30% for strains TA 98, TA 100 and TA 1535 and 30% for strain TA 1537)

Test concentrations with justification for top dose:

100, 333, 1000, 3333, or 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See Table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37 ºC
- Exposure duration: 48 hours at 37 ºC

NUMBER OF REPLICATIONS: At least five doses of each chemical were tested in triplicate, and repeat experiments were performed at least one week following the initial trial.

Evaluation criteria:

Revertant colonies were counted

Statistics:

None performed (revertant colonies were counted)

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no cytotoxicity when tested at levels ≤ 10,000 µg/plate which is higher than the limit concentration of 5,000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no cytotoxicity when tested at levels ≤ 10,000 µg/plate which is higher than the limit concentration of 5,000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

other: Revertant colony count did not double in the presence of 30% hamster liver microsomes. An adequate positive control response was observed for all other tested conditions.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Iospropanol was found negative with and without metabolic activation in this Ames test using strains TA97, TA98, TA100, TA1535 and TA 1537.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Upon contact with water or moisture (e.g. within mucous membranes) aluminium tri-isoproanolate hydrolyses immediately to isopropanol and aluminium 3+ cations (as hydroxide and oxyhydroxide). Hence, toxicity is determined by the toxicity of these two species.

In a study according to OECD 475 female rats (5/group) received aluminium oxide by oral at of 500, 1000 and 2000 mg Al2O3/kg bw in 1% Tween 80/doubly-distilled water (265, 529 and 1058 mg Al/kg bw) (Balasubramanyam 2009). A negative control group was treated orally with the vehicle. A positive control group received a single intraperitoneal dose of 40 mg/kg bw of cyclophosphamide. 500 well-spread metaphases (100 per animal) were analysed for each treatment 18 and 24 hours after the last dosing. Organ tissue was analyzed for aluminium content by ICP-MS. There was no indication of an effect of treatment on the mitotic index. Eighteen hours after the final dosing, the mean±sd total aberrations for the control, 265, 529 and 1058 mg Al/kg bw/day were 0.6±0.3, 0.6±0.3, 2.2±0.8, and 4.3±1.0, respectively. Statistical testing reported in the article indicated no significant differences for any treatment level of this group compared with the control group. The levels of aluminium in tissues show an increase with dose that does not reach statistical significance. A significant (p<0.01) increase in the Al2O3 content in faeces relative to the control was observed at 1058 mg Al/kg bw.

In a test according to OECD 474 (Balasubramanyam 2009) the frequency of micronuclei in polychromatic (immature) erythrocytes (MN-PCEs) in bone marrow was not significantly different from the vehicle control (1% Tween 80 in DDW) in any treated group indicating that cell death was not occurring as a result of treatment. At both 30 and 48 hours after administration of the last dose Al2O3 did not increase the number of MN-PCEs. The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls, but showed consistent increases in levels of Al in tissues and organs with increasing dose.

Isopropanol wat tested in an in vivo micronucleus test at 350, 1175 and 2500 mg/kg bw (iv in physicological saline). Toxicity (mortality, decreased body weight and macroscopic effects in the GI tract) was observed in the 2500 mg/kg bw group. Bone marrow was extracted from 10 mice/group (5 males/5 females) at 24, 48, and 72 hours after dosing. Isopropanol induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes (Ivett 1991).

Link to relevant study records

Reference

Reference 1

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:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

purity > 90%
particle size: 50-200 um

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 old
- Weight at study initiation: 90-100 g
- Diet: ad libitum. The feed was described as standard laboratory feed (wheat flour 2.5%; roasted Bengal gram flour 60%; skimmed milk powder 5%; casein 4%; refined ground oil 4%; salt mixture 4%; vitamin mixture 0.5%).
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture
- Volume dosed: No data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Al2O3 was 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:

14 days

Frequency of treatment:

acute exposure (expected to be daily)

Post exposure period:

CA assay: 18h, 24 h

Mitotic Index (MI): 18h, 24h

Al Level Study:
Urine and faeces: 48 h after (first?) dosing
Blood and tissues (liver, spleen, heart, kidneys and brain): on day 14 at sacrifice

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 265 mg/kg bw as Al

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 529 mg/kg bw as Al

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 1069 mg/kg bw as 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: 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.

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

CA Assay: Al2O3 - (50-200 μm)

Toxicity:

no effects

Remarks:

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

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MI: There was no significant reduction in MI at either sampling time for any of the dose groups compared with the vehicle control.

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

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

CA assay:

Total aberrations (excluding gaps)

 

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

18h: 0.6(±0.32)

24h: 0.5(±

0.42)

 

Al₂O₃- (50-200 μm)

18h:

25 mg Al:0.6±0.22 - ns

50 mg Al: 2.2±0.79 - ns

100 mg Al: 4.3±1.01 - ns

24h:

25 mg Al:0.5±0.31 - ns

50 mg Al: 1.2±0.51 - ns

100 mg Al: 5.5±0.31 - ns

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

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

Al Levels

Control (units±sd)

Whole blood   3±1 μg/g

Liver              6±2μg/g

Spleen            2±1μg/g

Heart             6±3 μg/g

Kidneys          9±4 μg/g

Brain              2±3 μg/g

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

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

 

Al₂O₃- (50-200 μm)”bulk”

The levels in tissues show an increase with dose that does not reach statistical significance. A significant (p<0.01) increase in the Al₂O₃content in faeces relative to the control was observed at 2000 mg/kg bw. The amounts of Al in tissues of the nanomaterial dosed animals were much higher than those found in animals dosed with the bulk material.

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.

Conclusions:

No significant increase in the number of aberrations. Therefore it can be concluded that Al2O3 does not exhibity clastogenic effects under the conditions of the test

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 1059 mg Al/kg bw. Three size fractions of aluminium oxide particles were examined: Al2O3 (30 nm; transmission electron microscopy (TEM) determined diameter, mean±sd - 39.85±31.33 nm), Al2O3 (40 nm; TEM diameter, mean±sd – 7.33±36.13 nm), and Al2O3-bulk (diameter 50 to 200μm). A negative control group was treated orally with the Tween 80/DDW vehicle.A positive control group received a single intraperitoneal dose of 40 mg/kg bw of cyclophosphamide. The assessment of CA in bone marrow cells was conducted in accordance with OECD Test Guideline #475 with the analysis of 500 well-spread metaphases (100 per animal) for each treatment 18 and 24 hours after the last dosing. Aneuploidy, polyploidy, gaps, breaks, minutes, acentric fragments and reciprocal translocations were counted. The mitotic index was determined on 1000 cells at both sampling times, slides were selected randomly and coded to blind analysts. Dose levels were determined using an acute oral toxicity study conducted in accordance with OECD Test Guideline #420. Organ tissue was analyzed for aluminium content by ICP-MS (inductively coupled mass spectrometry). The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. There was no indication of an effect of treatment on the mitotic index. 

Eighteen hours after the final dosing, 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. Statistical testing reported in the article indicated no significant differences for any treatment level of this group compared with the control group. The reported measurements of levels of Al in the urine and faeces sampled 48 hours after dosing and in tissues in samples taken on day 14 were reported as μg Al2O3/g wet tissue (clarified after contact with the author). Aluminium levels were elevated in all tissues in a dose-response manner with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The measurements do, however, show consistent increases inlevels of Al in tissues and organs with increasing dose.

 

Reference 2

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:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Principles of method if other than guideline:

purity > 90%
particle size: 50-200 um

GLP compliance:

not specified

Type of assay:

micronucleus assay

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 – 100 g
- Diet: ad libitum. The feed was described as standard laboratory feed (wheat flour 2.5%; roasted Bengal gram flour 60%; skimmed milk powder 5%; casein 4%; refined ground oil 4%; salt mixture 4%; vitamin mixture 0.5%).
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycles

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DDW-Tween 80 (1%) mixture
- Volume dosed: no data

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:

14 days

Frequency of treatment:

acute exposure (expected to be daily)

Post exposure period:

MN assay: 30h, 48h

Mitotic Index (MI): 18h, 24h

Al Level Study:
Urine and faeces: 48 h after (first?) dosing
Blood and tissues (liver, spleen, heart, kidneys and brain): on day 14 at sacrifice

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 265 mg/kg bw as Al

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 529 mg/kg bw as Al

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

as Al2O3; 1059 mg/kg bw as Al

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Substance: Cyclophosphamide
Justification: Known mutagen on list of acceptable positive control substances in the relevant OECD TGs.
Route of administration(s): intraperitoneal
Dose/concentration: 40 mg/kg bw, volume= 0.01mL/g bw

Tissues and cell types examined:

Rat bone marrow.

Details of tissue and slide preparation:

MN assay:
The authors stated that the measurements were made in accordance with OECD TG#474. A minimum of 4 slides were made for each animal for assessment of MN frequency and stained with Giemsa. Slides were coded. A total of 2000 PCEs (polychromatic erythrocytes) from all 4 slides were scored for MN.

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

Cytotoxicity (Mitotic Index):
The MI was determined on 1000 cells or more from randomly selected slides that were coded prior to scoring.

Evaluation criteria:

The criteria used for a positive response were not provided explicitly but the guidelines were cited.

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

Statistics:

The methods used were included in the article but were not well-described. The LSD-test mentioned in the article is more appropriate for particular planned comparisons and not for comparing several pairs of means. Testing for homogeneity of variances was also not mentioned. The results show some evidence of an increase in the magnitude of the standard deviation with dose for some endpoints.

Key result

Sex:

female

Genotoxicity:

negative

Remarks:

MN-Assay: Al2O3 - (50-200 μm)

Toxicity:

no effects

Remarks:

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

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MI: There was no significant reduction in MI at either sampling time for any of the dose groups compared with the vehicle control.

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

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

MN Assay:

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

 

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

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

 

Negative control (1% Tween 80)

MN-PCEs/2000 PCEs, mean ±sd

30h: 2.5 ±0.70

48h: 1.8 ±0.75

Al₂O₃- (50-200 μm)

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

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

30h:

25 mg Al: 1.9±0.73, ns

50 mg Al: 3.3±1.16, ns

100 mg Al: 5.9±1.71, ns

48h:

25 mg Al: 2.0±0.64, ns

50 mg Al: 4.2±1.07, ns

100 mg Al: 6.6±1.68, ns

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

 

Al Levels

Control (units±sd)

Whole blood   3±1 μg/g

Liver              6±2μg/g

Spleen            2±1μg/g

Heart             6±3 μg/g

Kidneys          9±4 μg/g

Brain              2±3 μg/g

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

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

 

Al₂O₃- (50-200 μm)”bulk”

The levels in tissues show an increase with dose that does not reach statistical significance. A significant (p<0.01) increase in the Al₂O₃content in faeces relative to the control was observed at 2000 mg/kg bw. The amounts of Al in tissues of the nanomaterial dosed animals were much higher than those found in animals dosed with the bulk material. (data not reported)

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.

Conclusions:

No significant increase in the number of micronucleated cells. Therefore it can be concluded that Al2O3 does not exhibity genotoxic effects under the conditions of the test
The genotoxicity results for 50 to 200 μm diameter particles (Al2O3-bulk) were not significantly different from those of the vehicle control.

Executive summary:

Balasubramanyam et al. (2009) also determined the frequency of micronuclei in polychromatic (immature) erythrocytes (MN-PCEs) in bone marrow according to OECD Test Guideline #474 (1997). The percentage of PCEs was not significantly different from the vehicle control (1% Tween 80 in DDW) in any treated group indicating that cell death was not occurring as a result of treatment. At both 30 and 48 hours after administration of the last dose, the 50 to 200 µm sized Al2O3-bulk particles did not increase the number of MN-PCEs. A Klimisch Score of 2 was assigned to the MN assay results from this study.

Aluminium levels were elevated in all tissues in a dose-response manner with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200 μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The measurements do, however, show consistent increases inlevels of Al in tissues and organs with increasing dose.

Balasubramanyam et al. (2009) 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 wereμg Al2O3/g wet tissue. Aluminium levels were elevated in all tissues in a dose-response manner with either of the nano-sized particulates. A particle size dependence of gastrointestinal absorption was apparent with lower levels of aluminium in the tissues reported for the larger 50 to 200μm diameter particles (Al2O3-bulk). The levels of Al in the Al2O3-bulk treated groups showed an increase but were not reported as significantly different from the controls. The measurements do, however, show consistent increases inlevels of Al in tissues and organs with increasing dose. Nano-sized materials were absorbed to a greater extent than Al2O3-bulk (50-200µm). The units used in the article for levels in urine (µg/mL) indicate Al concentrations in the urine of the control group to be 3-orders of magnitude higher than the normal range in humans (1-2 µg/L) (Krewski et al., 2007). The reliability of the aluminium measurements in tissues and urine is limited by the inconsistencies and lack of clarity in reporting. A Klimisch Score of 3 was assigned to the Al tissue measurements.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation. The publication is sufficiently detailed to allow a scientific evaluation of the results

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

- 1000 polychromatic erythrocytes were evaluated per animal, rather than 2000

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from Harlan Sprague-Dawley, Inc., Frederick, MD
- Age at study initiation: 8 to 11 weeks old
- Assigned to test groups randomly: yes, under following basis: by a computer generated randomization program
- Housing: 5 mice/cage
- Diet (e.g. ad libitum): Purina Certified Laboratory Chow #5002 ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Quarantined for seven days before being placed on study

ENVIRONMENTAL CONDITIONS
- Temperature: Reported in the study to be 72 ± 6 ºF (approximately 22.2 ºC)
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 hours: 12 hours

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.9% sodium chloride

Details on exposure:

Thirty randomly assigned mice/group (15 males/15 females) were treated with isopropanol dissolved in 0.9% sodium chloride at dose levels of 0 (vehicle control), 350, 1173 and 3500 mg isopropanol/kg bw by intraperitoneal injection. Mice were observed for toxic symptoms and/or mortalities immediately after dosing and twice daily for the duration of the assay.

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Single exposure

Post exposure period:

None

Remarks:

Doses / Concentrations:
350 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1173 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2500 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
3500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 80 mg/kg bw

Tissues and cell types examined:

Bone marrow was extracted from 10 mice/group (5 males/5 females) at 24, 48, and 72 hours after dosing. An additional group of 10 mice (5 males/5 females) was treated with 3500 mg/kg bw and held to ensure that 10 mice were available for bone marrow extraction at each interval. These mice were scheduled for use only if mortalities occurred in the primary dose group. As needed, mice were randomly selected from this secondary group for bone marrow extraction and unused mice were euthanized at the completion of the trial. Bone marrow for the positive control group was harvested at 24 hours. Terminal body weights were collected on all animals prior to euthanization for bone marrow harvest.

Details of tissue and slide preparation:

At the appropriate harvest time, the animals were euthanized with CO2 and the adhering soft tissue and epiphyses of both tibias were removed. The marrow was flushed into a centrifuge tube (one tube for each animal) with 3 mL fetal calf serum. Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol and stained in May-Gruenwald solution followed by Giemsa. After being air-dried, the slides were coverslipped using Depex mounting medium. The coded slides were then scored blind for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

Statistics:

ANOVA followed by Tukey's

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See remarks section.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

All animals in the 3500 mg/kg bw dose group became prostrate after dosing. Within 22 hours after dosing, 35 of the 40 animals dosed at 3500 mg/kg had expired. Due to this toxicity, the 3500 mg/kg bw dose level was eliminated from the study.

Immediately after dosing, the animals receiving 2500 mg/kg became prostrate. Approximately four hours after dosing, the animals were languid with squinted eyes. The following morning, approximately 23 hours after dosing, one male (#8371) was languid with squinted eyes. This animal also exhibited dyspnea approximately 30 hours after dosing. All other animals appeared normal; however, within 46 hours of dosing three animals (male #8371; female #’s 8319 and 8354) were found dead. Another female (#8369) expired approximately 53 hours after dosing and several other animals were languid. On the third morning, approximately 70 hours after dosing, two males (#’s 8326 and 8405) were found dead. All remaining test article dosed animals had rough haircoats and this condition remained at the 72 hour harvest time. A gross necropsy was performed on all animals which expired during the observation period. Male #8371 had a moderate amount of clear yellow fluid in the trachea and thoracic cavity and an irregular black stomach mucosa. Female #8369 also had a moderate amount of a clear orange fluid in the thoracic cavity. The other necropsied animals had moderate to significant distension of the stomachs or colons, two with an abnormally thin fluid content. No other abnormalities were noted in the animals examined. The terminal body weight range of the animals harvested in this trial of the micronucleus assay was 28.1 - 37.4 grams for males and 19.0 - 26.9 grams for females. Terminal body weight gains were significantly lower at the 48 and 72 hour sacrifice intervals in mice treated with 2,500 mg/kg bw compared to corresponding vehicle control body weight gains indicating that there was a test article related reduction in body weights.

The test article, isopropanol, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 3.24% ± 0.84% and 1.22% ± 0.19% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 1).

The test article, isopropanol, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.56% ± 0.42% and 1.66% ± 0.39% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 2).

Conclusions:

Interpretation of results (migrated information): negative
The test material, isopropanol, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on available data the substance is not considered being genotoxic/mutagenic and thus does not require classification according to DSD (Directive 67/548/EEC) or CLP (Regulation EC No 1272/2008) for mutagenicity.