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Genetic toxicity in vitro

Description of key information

The three key studies are:

- OECD 471 GLP Salmonella/E.coli bacterial mutation assay

- OECD 476 GLP mammalian cell L5178Y mutation assay

- OECD 487 GLP human lymphocyte chromosome aberration (micronucleus) assay in mammalian cells.

All three are GLP, Klimisch grade 1 studies and conducted according to the relevant OECD test guidelines and meet the Annex VIII test requirements of REACH. All three studies provide clear negative (non genotoxic) results.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 11 to 22, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test guideline No. 471 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Experiment-1
Dose range finding test (without and with 5 % (v/v) S9-mix): 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in TA 100 and WP2uvrA strains.
Main test (without and with 5 % (v/v) S9-mix): 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537 and TA 98 strains.

Experiment-2 (without and with 10 % (v/v) S9-mix): 33, 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537, TA 98 and TA 100 strains; 100, 333, 1000, 3330 and 5000 µg/plate in WP2uvrA strain.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: Test substance was dissolved in Milli-Q water.
- The stock solution was filter (0.22 μm)-sterilized. Test substance concentrations were used within 2.5 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h at 37.0 ± 1.0 °C

NUMBER OF REPLICATIONS: 3 plates/dose for Experiment 1 and 2

DETERMINATION OF CYTOTOXICITY
- Method: Test material toxicity was determined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

OTHER:
Colony counting: - The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these could be counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.
Evaluation criteria:
- A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
- A test substance is considered positive (mutagenic) if:
(a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
(b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
- The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
- No formal hypothesis testing was done.
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: Soluble in water.
- Precipitation: No test material precipitation was observed on the plates at the start or at the end of the incubation period.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: The bacterial background lawn was not reduced at any of the concentrations tested. The number of revertants of TA100 was moderately decreased at 5000 µg/plate in the absence of S9-mix. In the presence of S9-mix, no biologically relevant decrease in the number of revertants was observed in tester strain WP2uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 3330 µg/plate and above and with S9: 3330 µg/plate and above
TA1537: without S9: 1000 µg/plate and above and with S9: 3330 µg/plate and above
TA98: without S9: 333 µg/plate and above and with S9: 5000 µg/plate
TA100: without S9: 3330 µg/plate and above and with S9: 5000 µg/plate

See the “Attached background material” section for further details
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test material at the following concentrations both in the presence and absence of metabolic activation system (rat liver S9-mix) using the plate incorporation method in two independent experiments.

Experiment-1: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in TA 100 and WP2uvrA strains; 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537 and TA 98 strains, without and with 5 % (v/v) S9-mix).

Experiment-2: 33, 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537, TA 98 and TA 100 strains; 100, 333, 1000, 3330 and 5000 µg/plate in WP2uvrA strain, without and with 10 % (v/v) S9-mix)

Vehicle and positive control groups were also included in mutagenicity tests.

The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Toxicity was observed in tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation in two independently repeated experiments.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 30 to September 07, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline No. 476 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source: American Type Culture Collection (ATCC), Manassas, USA
- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- R5: Basic medium, supplemented with 5 % (v/v) heat-inactivated horse serum.
- R10: Basic medium, supplemented with 10 % (v/v) heat-inactivated horse serum.
- R20: Basic medium, supplemented with 20 % (v/v) heat-inactivated horse serum.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: Yes; the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
- Dose range finding test:
3 h treatment, with and without S9-mix: 11, 36, 109, 363 and 725 µg/mL
24 h treatment, without S9-mix: 11, 36, 109, 363 and 725 µg/mL
- First mutagenicity test:
3 h treatment, without and with 8 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL
- Second mutagenicity test:
3 h treatment, with 12 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 μg/mL
24 h treatment, without S9-mix: 1, 3, 10, 33, 100, 333, 400, 450, 500, 550, 600 and 666 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium.
- Test material preparation: Test material at concentrations of 12 mg/mL and higher was dissolved in RPMI 1640 (Hepes buffered medium in the dose range finding test and the first mutation experiment. Test material was suspended at concentrations between 1.2 and 4.3 mg/mL in RPMI 1640 medium whereas at concentration of 0.4 mg/mL and lower, test material was again soluble in RPMI 1640 medium. In the second mutation experiment, at concentrations of 40 mg/mL and higher test material was dissolved in RPMI 1640. At concentrations between 0.4 and 12 mg/mL the test substance was suspended in RPMI 1640 medium and at concentrations of 0.12 mg/mL and lower the test substance was again soluble in RPMI 1640 medium. The stock solutions were filter (0.22 μM)-sterilized and test concentrations were used within 1.5 h after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix: Methylmethanesulfonate at 15 and 5 μg/mL for a 3 and 24 h treatment period, respectively.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix: 7.5 μg/mL of cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: RPMI 1640 medium

DURATION
- Exposure duration:
Dose range finding test: 3 h with and without S9-mix and 24 h without S9-mix.
First mutagenicity test: 3 h with and without 8 % (v/v) S9-mix.
Second mutagenicity test: 3 h with 12 % (v/v) S9-mix and 24 h without S9-mix.
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent):11 or 12 days

SELECTION AGENT (mutation assays): Selective medium consisted of R20 containing 5 μg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS:
- Dose range finding test: Single culture/dose
- Mutagenicity test: Single cultures/dose for test item and positive controls, 2 cultures for vehicle control

NUMBER OF CELLS EVALUATED: 1 and 2000 cells/well plated for assessing cloning efficiency (CE day2) and mutation frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

- Other:
Calculation of the mutation frequency: The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
Mutation frequency (MF) = {-ln P(0) / number of cells plated per well} / CEday2 x 10^6
Small and large colony mutation frequencies were calculated in an identical manner.
Evaluation criteria:
- A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test substance is considered negative (not mutagenic) in the mutation assay if:
(a) None of the tested concentrations reaches a mutation frequency of MF (controls) + 126.
(b) The results are confirmed in an independently repeated test.
- In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative / solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (Moore et al., 2006).
Statistics:
None
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Test material precipitated in the exposure medium at concentrations of 333 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES: After 3 h of treatment without S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 725 μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 66 % at the highest test substance concentration of 725 μg/mL compared to the relative suspension growth of the solvent control.
After 24 h of treatment without S9-mix, the relative suspension growth was 45 % at the test substance concentration of 363 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 725 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

Second mutagenicity test: In the absence of S9-mix, the dose levels of 1 to 100 μg/mL showed no cytotoxicity. Therefore, the dose level of 100 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 333 and 400 μg/mL and 450 to 550 μg/mL showed similar cytotoxicity. Therefore, the dose levels of 400, 450 and 550 μg/mL were not regarded relevant for mutation frequency measurement. The relative total growth of the highest test substance was reduced by 90% compared to the total growth of the solvent controls.
In the presence of S9-mix, no toxicity was observed and all dose levels were evaluated.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See the attached document for information on tables of results.

Conclusions:
Under the test conditions, test material is not mutagenic at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, in the absence and presence of S9-mix.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline No. 476 and in compliance with GLP, L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to test material at the following concentrations:

- Dose range finding test:
3 h treatment, with and without S9-mix: 11, 36, 109, 363 and 725 µg/mL
24 h treatment, without S9-mix: 11, 36, 109, 363 and 725 µg/mL
- First mutagenicity test:
3 h treatment, without and with 8 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL
- Second mutagenicity test:
3 h treatment, with 12 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 μg/mL
24 h treatment, without S9-mix: 1, 3, 10, 33, 100, 333, 400, 450, 500, 550, 600 and 666 µg/mL

Vehicle and positive control groups were also included in each mutation test.

In the dose range finding test, after 3 h of treatment without S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 725 μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 66 % at the highest test substance concentration of 725 μg/mL compared to the relative suspension growth of the solvent control. After 24 h of treatment without S9-mix, the relative suspension growth was 45 % at the test substance concentration of 363 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 725 μg/mL. Test material precipitated in the exposure medium at concentrations of 333 μg/mL and above. In the mutagenicity test, the relative total growth was reduced by 90 % at 666 μg/mL compared to the total growth of the solvent controls at 24 h treatment without S9-mix. Test material did not induce a significant increase in the mutation frequency at any dose level either with or without metabolic activation in two independently repeated experiments. In all tests the concurrent vehicle and positive control were within acceptable ranges.

Under the test conditions, test material is not mutagenic at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, in the absence and presence of S9-mix.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 05 to May 04, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline No. 487 (draft) without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Draft proposal for a new Guideline No. 487: In Vitro Mammalian Cell Micronucleus Test (November 2, 2009).
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
1.8 % (v/v) S9: Rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Dose range-finding test: 0, 10, 33, 100, 333 and 1000 μg/mL, with and without S9-mix (3 h exposure time, 27 h harvest time); 0, 10, 33, 100, 333, 1000 and 3330 μg/mL, without S9-mix (24 h exposure time, 24 h harvest time)
First cytogenetic assay: 0, 100, 333 and 1000 μg/mL, with and without S9-mix (3 h exposure time, 27 h harvest time)
Cytogenetic assay 1A: 0, 30, 100, 200, 300 and 700 μg/mL, with S9-mix (3 h exposure time, 27 h harvest time)
Second cytogenetic assay: 0, 50, 100, 500, 1000, 1250, 1500 and 1750 μg/mL, without S9-mix (24 h exposure time, 24 h harvest time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Test material preparation: Test material was dissolved in RPMI 1640 medium at concentrations 20 mg/mL and above. At concentrations of 10 mg/mL and below test material precipitated in RPMI 1640 medium. Test material concentrations were used within 2.5 h after preparation and filter (0.22 μm)-sterilized.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C: 0.25 μg/mL (3 h exposure), 0.15 μg/mL (24 h exposure); Colchicine: 0.1 μg/mL (3 h exposure), 0.05 μg/mL (24 h exposure)
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide: 15 μg/mL for 3 h exposure
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.

CELL CULTURE: Lymphocytes (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 h.


DURATION
- Exposure duration: Dose range finding test: 3 h (with and without S9-mix) and 24 (without S9-mix); First cytogenetic assay: 3 h (with and without S9-mix); Second cytogenetic assay: 24 h (without S9-mix).
- Fixation time (start of exposure up to fixation or harvest of cells): Dose range finding test: 27 h (with and without S9-mix) and 24 (without S9-mix); First cytogenetic assay: 27 h (with and without S9-mix); Second cytogenetic assay: 24 h (without S9-mix).

SPINDLE INHIBITOR (cytogenetic assays): 5 µg/mL of Cytochalasin B

STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution in water for 10-30 minutes.

NUMBER OF REPLICATIONS: Two cultures/dose

NUMBER OF CELLS EVALUATED:
- Cytotoxicity assessment: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
- At least 1000 binucleated cells per culture were examined by light microscopy for micronuclei. In addition, 1000 mononucleated cells per culture were scored for micronuclei separately.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)
- CBPI was calculated using formula below:
CBPI = {(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)}/ Total number of cells
- Cytostasis (%) = 100-100 {(CBPIt – 1)/ (CBPIc-1)}; t = test substance or control treatment culture, c = vehicle control culture


OTHER:
1. The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
2. The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
3. The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.
Evaluation criteria:
- A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
- A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, one sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
- The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
- The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics. If p<0.05, the number of micronucleated cells in the test group is considered to be significantly different from the control group at the 95% confidence level.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Yes
- Precipitation: Test material precipitated in the culture medium at 700 µg/mL, with S9-mix (in cytogenetic assay 1A) and at 1000, 1250, 1500 and 1750 µg/mL, without S9-mix (in second cytogenetic assay).

RANGE-FINDING/SCREENING STUDIES: Test material precipitated in the culture medium at a concentration of 1000 μg/mL. No toxicity was observed up to the concentrations of 1000 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: See the attached document for tables of results.

OTHERS:
- The highest concentration analysed was selected based on toxicity (24 h exposure time), cytokinesis-block proliferation index of 55 ± 5% or on the solubility of the test substance in the culture medium (3 h exposure time).
Remarks on result:
other: other: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/1: Results summary

Treatment

Concentration

(μg/mL)

Cytostasis

(%)

Number of mononucleated cells with micronuclei

Number of binucleated cells with micronuclei

1000

1000

2000

1000

1000

2000

A

B

A + B

A

B

A + B

3 h exposure,

27 h harvest,

- S9-mix

0

0

1

0

1

3

2

5

100

8

0

1

1

3

1

4

333

15

0

1

1

5

4

9

1000#

24

0

2

2

2

4

6

0.25 MMC-C

41

4

2

6*

47

40

87***

0.1 Colch

84

61

61

122***

36

30

66***

3 h exposure,

27 h harvest ,

+ S9-mix

0

0

1

0

1

1

0

1

100

3

0

0

0

1

2

3

300

16

0

0

0

2

0

2

700#

22

2

1

3

4

1

5

15 CP

65

1

1

2

24

30

54***

24 h exposure, 24 h harvest,   

 -S9-mix

 

0

0

0

0

0

2

0

2

100

4

0

0

0

1

1

2

1000

25

0

0

0

2

0

2

1500#

44

0

0

0

1

0

1

0.15 MMC-C

54

6

4

10***

20

16

36***

0.05 Colch

100

49@

55$

104***

1##

0$$

1

 

Keys:                                                                                                                    

Duplicate cultures are indicated by A and B.

* Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

# Test material precipitated in the culture medium.

@ Number of micronucleated cells per 587 mononucleated cells

$ Number of micronucleated cells per 624 mononucleated cells

## Number of micronucleated cells per 69 binucleated cells

$$ Number of micronucleated cells per 89 binucleated cells

MMC-C: Mitomycin C

Colch: Colchicine

CP: Cyclophosphamide

Conclusions:
Under the test conditions, test material is not clastogenic or aneugenic in human lymphocytes in the absence and presence of S9-mix.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test material in the presence and absence of a metabolic activation system (1.8 % (v/v) S9-mix). Dose range finding test was performed to select the appropriate dose levels for the cytogenetic assays. In the first cytogenetic assay, test material was tested up to 1000 and 700 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence and presence of S9-fraction, respectively. In the second cytogenetic assay, test material was tested up to 1500 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Cytochalasine B (5 μg/mL) was added to the cells simultaneously with the test substance. The cells were then treated with a hypotonic solution, fixed, stained and examined for toxicity and micronuclei. Vehicle and positive controls were also included in the study.

The number of mono- and binucleated cells with micronuclei found in the vehicle control cultures was within the laboratory historical control data range. The positive control chemicals produced a statistically significant increase in the number of cells with micronuclei, demonstrating the sensitivity of the test system. Test material precipitated in the culture medium at 700 µg/mL and above concentrations. Test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Under the test conditions, test material is not clastogenic or aneugenic in human lymphocytes in the absence and presence of S9-mix.

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

Genetic toxicity in vivo

Description of key information

A GLP, Klimisch Grade 1 in vivo micronucleus study on aluminium hydroxide has been used as a read-across study (K2 in this case) in support of the three key in vitro studies. It is considered that a read-across from aluminium hydroxide to aluminium chloride is justified and robust because the two salts have similar levels of absorption of aluminium after oral dosing (Priest., K 2010 - see toxicokinetics section) The RA in vivo study provided a negative conclusion for in vivo genotoxicity, which is in agreement with the Key in vitro study results on aluminium chloride.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
5 Jan 2010 – 4 Feb 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP - Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: range-finder experiment: 6-10 weeks; micronucleus experiment: 8 weeks
- Weight at study initiation: range-finder experiment: 181-190 g; micronucleus experiment: 217-260 g
- Housing: The rats were housed in an air-conditioned room (15 air exchanges/hour) in groups, up to six per group, “in cages with the 'Code of practice for the housing and care of animals used in scientific procedures”
- Diet (e.g. ad libitum): ad libitum access to SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham)
- Water (e.g. ad libitum): Mains water ad libitum via water bottles
- Acclimation period: at least 5 days

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

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

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


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

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

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

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

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

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

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

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

The laboratory historical positive control ranges are also presented

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

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

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

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

 

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

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

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

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

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

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

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

(dose)                                                           

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

0              49.35             2.67                   0.13 (0.10)

500           58.55             2.33                   0.12 (0.09)

1000         53.08             2.83                   0.14 (0.09)

2000         54.82             2.50                   0.13 (0.06)

PC*          46.13            55.50                  2.78 (1.60)

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

*PC-positive control

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

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

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

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

Additional information

Key Studies

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test material at up to the maximum recommended dose level, both in the presence and absence of metabolic activation system (rat liver S9-mix) using the plate incorporation method in two independent experiments.Vehicle and positive control groups were also included in mutagenicity tests. The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Toxicity was observed in tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation in two independently repeated experiments. Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coliWP2uvrA.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test material in the presence and absence of a metabolic activation system (1.8 % (v/v) S9-mix). Dose range finding test was performed to select the appropriate dose levels for the cytogenetic assays. In the first cytogenetic assay, test material was tested up to 1000 and 700 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence and presence of S9-fraction, respectively. In the second cytogenetic assay, test material was tested up to 1500 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Cytochalasine B (5 μg/mL) was added to the cells simultaneously with the test substance. The cells were then treated with a hypotonic solution, fixed, stained and examined for toxicity and micronuclei. Vehicle and positive controls were also included in the study. The number of mono- and binucleated cells with micronuclei found in the vehicle control cultures was within the laboratory historical control data range. The positive control chemicals produced a statistically significant increase in the number of cells with micronuclei, demonstrating the sensitivity of the test system. Test material precipitated in the culture medium at 700 µg/mL and above concentrations. Test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Under the test conditions, test material is not clastogenic or aneugenic in human lymphocytes in the absence and presence of S9-mix.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline No. 476 and in compliance with GLP, L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to test material with a range of concentrations. Vehicle and positive control groups were also included in each mutation test. In the dose range finding test, after 3h of treatment without S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 725 μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 66 % at the highest test substance concentration of 725 μg/mL compared to the relative suspension growth of the solvent control. After 24 h of treatment without S9-mix, the relative suspension growth was 45 % at the test substance concentration of 363 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 725 μg/mL. Test material precipitated in the exposure medium at concentrations of 333 μg/mL and above. In the mutagenicity test, the relative total growth was reduced by 90 % at 666 μg/mL compared to the total growth of the solvent controls at 24 h treatment without S9-mix. Test material did not induce a significant increase in the mutation frequency at any dose level either with or without metabolic activation in two independently repeated experiments. In all tests the concurrent vehicle and positive control were within acceptable ranges. Under the test conditions, test material is not mutagenic at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, in the absence and presence of S9-mix.

Read-Across and Weight of Evidence/Disregarded Studies

Two GLP, in vivo micronucleus key studies on aluminium hydroxide and dialuminium chloride pentahydroxide (Locron P) have been used as a read-across study in support of the three key in vitro studies. It is considered that a read-across from aluminium hydroxide or dialuminum cjloride pentahydroxide to aluminium sulphate is justified and robust because the three salts have similar levels of absorption of aluminium after oral dosing (Priest., K 2010 - see toxicokinetics section) The RA in vivo studies provided a negative conclusion for in vivo genotoxicity, which is in agreement with the Key in vitro study results on aluminium sulphate.

A total of six in vitro studies and 2 in vivo study have been included as weight of evidence or disregarded studies. Some of these studies provided negative results that were in agreement with the conclusions of the Key and RA studies. However, some of the studies also provided contrary positive conclusions. But none of the studies reported as positive were GLP and thus were classified as Klimisch grade 3 or 4. In addition, these studies were poorly executed and inaequately reported. The dose levels used were not clearly justified and the time-points used were different from the guideline recommendations. An expert review revealed that none of these studies was considered to be sufficiently reliable to add any significant weight against the strength of the Key and RA studies.

The dossier includes other study summaries of studies published in the literature. These are of mostly poor quality and are non-GLP with many deviations from recoqnised test methods and good scientific practise. Some of these studies are reported by the authors as showing positive (genotoxic) results. However, none of the data are reliable and in a weight of evidence approach they carry little weight in comparison to the Key studies. Particular note is made of the publication by Paz, et al., 2017, included as a RSS, because it is a recent publication and claims to have been performed according to OECD 474. However, there are multiple reasons why this publication is considered to be scientifically unreliable and why the data are not plausible. In particular, significant histopathological effects on the stomach, kidney and liver are reported following a single oral dose of aluminium chloride, using relatively low dose levels. Such effects were not observed following repeat dosing at higher concentrations in high quality GLP studies, which indicates that the study is unreliable and none of the data may be accepted as valid.

Oxidative Mode of Action

There are many publications in the literature that propose an oxidative genotoxic mode of action for aluminium chloride and other salts of aluminium. Aluminium is generally accepted as being redox inert although some theoretical conditons predict a possible interaction between aluminium and superoxide anion, but only under anhydrous conditions.There are literature reports that aluminium soluble salts induce reactive species in vitro (e.g. brain, lymphocytes) and in repeated-dose toxicity studies in vivo (e.g. brain, blood, liver) in rabbits, mice and rats (Abd-Elghaffar et al., 2007, Candan et al., 2008, Prakash et al., 2009, Kaur et al., 2015, Lakshmi et al., 2015; Singh et al., 2015, Rani et al., 2015, Sood et al., 2015, Waly et al., 2014,). Several studies report, after administration of AC, alteration of the antioxidative system (superoxide dismutase, glutathione peroxidase, catalase), increase of reactive species (nitrite, nitrate, oxidized dichlorofluoresceine) and lipid peroxidation (increase in malondialdehyde, 4- hydroxyalkenals, thiobarbituric acid reactive substances). In these studies, this mechanism has been 'confirmed' experimentally by co-administration of antioxidants, which abrogated or reduced the effects of aluminium compounds on oxidative stress. However, all of the reports listed in the paragraph above are primarily interested in demonstrating that particular co-administrated substances (selenium, rifampicin, N-acetyl cysteine, diltazem, melatonin, tannic acid, curcumin, extracts of allium cepa, lazaroids and mushroom extract) may ameliorate the presumed oxidative toxicity and consequent neurotoxic effects of aluminium. However, only one of the studies (Waly et al., 2014) measured target tissue levels of aluminium to confirm exposure and they reported control levels of Al much higher than that of other workers (e.g., Baydar et al., 2002). Furthermore, pharmacokinetic studies on aluminium chloride have shown that oral absorption levels are so low that a significant period of low aluminium drinking water and feed is required to demonstrate uptake (Notox, 2010). None of these studies utilised a pre-study aluminium wash-out period. All of the studies reported neurotoxic effects in terms of behavioural performance deficiencies. However, no such neurotoxic effects were observed in the GLP, OECD studies such as the OECD 422 on AlCl basic. Furthermore, the study of Baydar et al., 2002 reported no changes in behavioural measures of neurotoxicity even though they measured higher levels of Al in brain tissue than control. It should be noted that Baydar used a high dose (200 mg/kg bw/day for 8 weeks) that was at least twice as high and for a longer duration than used in any of the studies quoted above. Also, the rigorous neurodevelopmental toxicity study by Poirier, 2011, showed no neurotoxicity effects after exposure throughout development and to 364 days of treatment.The behavioural-based evidence for neurotoxicity in the reports listed above is unreliable, because the behavioural responses observed are not reproduced in all studies, and, in particular, they werenot observedin the highly reliable GLP studies performed in laboratories, which are skilled in making such observations. In these studies, exposure levels anddurations were generally higher than those in the studies quoted above. If neurotoxicity is not reliably observed, then the observations used to support the explanation for such effects must also be unreliable.The modes of action and target molecules vary between the studies (as described above) to such an extent that it is difficult to accept that a common mode of action has been demonstrated. Furthermore, whilst many of the reports presented evidence that treatment with anti-oxidants resulted in reduced neurotoxic and oxidative effects, none of the studies included true oxidative positive controls. So, the evidence in these studies for an oxidative MoA is circumstantial at best. Furthermore, the GLP, OECD in vitro mammalian cell assays (key studies) are perfectly capable and sensitive to detect an oxidative mode of action, should one exist. The fact that no positive effects was observed in the key studies may be taken as strong evidence that AlCl is not genotoxic and does not have an oxidative mode of action (see attached document).

Endpoint Conclusion:

No adverse effect observed (negative).

References:

Abd-Elghaffar SKH, El Sokkary GH, Sharkawy AA (2007). Aluminum-induced neurotoxicity and oxidative damage in rabbits: protective effect of melatonin. Biogenic Amines, 21(4):225–240.

 

Candan N, Tuzmen N.Very rapid quantification of malondialdehyde (MDA) in rat brain exposed to lead, aluminium and phenolic antioxidants by high-performance liquid chromatography-fluorescence detection. Neurotoxicology. 2008 Jul;29(4):708-13.

 

Kaur P and Sodhi RK (2015). Memory recuperative potential of rifampicin in alumiium chloride-induced dementia: role of pregnane X receptors. Neuroscience 288 (2015) 24- 36.

 

Lakshmi BVS, M Sudhakar, KS Prakash Protective effect of selenium against aluminum chloride-induced Alzheimer's disease: behavioral and biochemical alterations in rats. Biol Trace Elem Res (2015) 165: 67.

 

Prakash A, Kumar A (2009). Effect of N-acetyl cysteine against aluminium-induced cognitive dysfunction and oxidative damage in rats. Basic&clinical pharmacology&toxicology, 105, 98-104.

POIRIER, H. SEMPLE, J. DAVIES, R. LAPOINTE, M. DZIWENKA,c M. HILTZ AND D. MUJIBI (2011).Double-blind, vehicle-controlled randomized twelve month neurodevelopmental toxicity study of common aluminum salts in the rat. Neurosciences 193, p 338 -362.

Rani A Neha, Sodhi R, Kaur A. Protective effect of a calcium channel blocker »diltiazem » on aluminium chloride-induced dementia in mice. Naunyn-Schmiedeberg's Arch Pharmacol. DOI 10.1007/s00210-015-1148-8.

 

Singh T, Goel RK. Neuroprotective effect of allium cepa L. in aluminium chloride induced neurotoxicity. Neurotoxicology 49 (2015) 1-7.

 

Sood PK, Verma S, Nahar U, Nehru B. Neuroprotective role of Lazaroids against aluminium chloride poisoning. Neurochem Res (2015) 40:1699-1708.

 

Waly MI and Guizani N. Antioxidant potential properties of mushroom extract (agaricus bisporous) aginst aluminium-induced neurotoxicity in rat brain. Pakistan Journal of biological sciences 17 (9):1079-1082, 2014.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

The Key studies show no positive results for mutagenicity or genotoxicity. In a weight of evidence approach the supporting studies are considered not to change the conclusion taken from the key studies.

Based on the available data, the substance is not classified for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.