Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

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

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-March-2010 to 11-March-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 33, 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: 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.
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 if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
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.
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 3330 μg/plate and above in the absence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our 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: 1000 µg/plate and above and with S9: 3330 µg/plate and above
TA1537: without S9: 1000 µg/plate and above and with S9: 1000 µg/plate and above
TA98: without S9: 1000 µg/plate and above
TA100: without S9: 3330 µg/plate and above and with S9: 5000 µg/plate

Conclusions:
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative 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.

Under the test conditions, the NOTOX substance 202029/A is not mutagenic in the presence and absence of metabolic activation in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and in Escherichia 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; 33, 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 all strains, 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. In tester strain TA100, toxicity was only observed at dose levels of 3330 and 5000 μg/plate in the absence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Cytotoxicity was observed in all tester strains except in tester strain TA98 in the presence of S9-mix. In the second experiment, cytotoxicity was observed in all tester strains except in the tester strains TA98 in the presence of S9-mix and in WP2uvrA 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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-Jul-2010 to 07-Sep-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
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):
- 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).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 11, 36, 109, 363 and 725 µg/mL
Without S9-mix, 24 hours treatment: 11, 36, 109, 363 and 725 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1, 3, 10, 33, 100, 250, 333 and 500 µg/mL
With S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium: RPMI 1640 (Hepes buffered medium (Dutch modification)
- Justification for choice of solvent/vehicle: Test compound was soluble in culture medium and culture medium has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 : 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9 : 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

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

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.
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
strain/cell type: L5178Y/TK+/-3.7.2C
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested beyond the limit of the solubility
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
strain/cell type: L5178Y/TK+/-3.7.2C
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 250 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- In the absence of S9, toxicity was only observed at dose levels of 109 µg/mL and above, 24 hours treatment.
- In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level (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:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was only reduced by 72% compared to the total growth of the solvent controls after the 24 hours treatment period.

In the absence of S9-mix (3 hours treatment) and in the presence of S9-mix (both experiments), no toxicity was observed up to and including the highest tested dose level of 333 µg/ml .
Conclusions:
Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

Under the test conditions, the NOTOX substance 202029/A is not mutagenic in the TK locus of L5178Y cells in the presence and absence of metabolic activation.
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, 250, 333 and 500 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, the relative suspension growth was 77% at 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 76 % 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 14 % at the test substance concentration of 725 μg/mL compared to the relative suspension growth of the solvent control. Test material precipitated in the exposure medium at concentrations of 363 μg/mL and above in all experimental conditions.

In the first mutagenicity test, the relative total growth was 84% and 103% at 333 μg/mL compared to the total growth of the solvent controls at 3 h treatment without and with 8% S9-mix, respectively. Precipitation was observed at the highest dose tested in both experimental conditions, i.e. 333 µg/mL. In the second mutagenicity test, the relative total growth was 28% at 500 µg/mL and 103% at 333 μg/mL compared to the total growth of the solvent controls for the 24 h treatment without S9 -mix and for the 3 h treatment with 12% S9-mix, respectively. Precipitation was recorded from 250 µg/mL and at 333 µg/mL for the 24 h treatment without S9-mix and for the 3 h treatment with S9-mix, respectively.

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:
05-mar-2010 to 03-may-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
- Source of cells
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture 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.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hr exposure; 27 hr fixation: 10, 33, 100, 333 and 1000 µg/mL
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 33, 100, 333, 1000 and 1105 µg/mL
First experiment
Without S9-mix, 3 h exposure time, 27 h fixation time: 100, 300 and 600 µg/mL
With S9-mix, 3 h exposure time, 27 h fixation time: 300, 500 and 600 µg/mL
Second experiment
Without S9-mix, 24hr exposure; 24 hr fixation: 100, 300 and 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Exposure medium (RPMI 1640 medium)
- Justification for choice of solvent/vehicle: Test compound was soluble in the exposure medium, this solvent has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 6: 0.25 µg/mL for a 3 h exposure period, 0.15 µg/mL for a 24 h exposure period
Positive control substance:
other: colchicine: 0.1 µg/ml for a 3 hours exposure period and 0.05 µg/ml for a 24 hours exposure period
Remarks:
without S9
Positive control substance:
cyclophosphamide
Remarks:
with S9 : 0.1 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9: 3 hr treatment, 24 hours recovery/harvest time
Continuous treatment
Without S9: 24 hours treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CBPI index).


OTHER:
- Interphase cells (mono and bi-nucleated cells) were analysed microscopically for the presence of micronuclei
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.
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.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 600 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed at the dose level of 1000 µg/ml in the presence of S9, 3 hr treatment/27 hr fixation

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of mono and binucleated cells with micronuclei observed in 2000 cells found in the solvent and positive control cultures was within the laboratory historical control data range.


The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range, except in the presence of S9-mix. Although the number of binucleated cells with miconuclei was above the historical control data range in the presence of S9-mix, the number of binucleated cells with micronuclei was less than 10. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:
The test substance 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, the NOTOX substance 202029/A is not clastogenic or aneugenic in human lymphocytes in the presence and absence of metabolic activation.
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 at 100, 300 and 600 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence and presence of S9-fraction. The experiment with S9 -miw was repeated because precipitate interfered with the scoring for the cytokinesis-block proliferation index; the concentrations of 100, 300, 400, 500 and 600 µg/mL were tested. In this experiment, no precipitate was observed and the concentrations 300, 500 and 600 µg/mL were selected for scoring of micronuclei. In the second cytogenetic assay, test material was tested at 100, 300 and 600 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. For this experiment, 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 600 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

Referenceopen allclose all

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:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common precursor(s)/breakdown product(s): Al3+ is the substance of biological interest and that the toxicological effects of Aluminium sulphate can be attributed exclusively to Al3+.

2. SOURCE AND TARGET CHEMICAL(S)
Source substance: Aluminium hydroxide (CAS No 21645-51-2)
Target substance: Aluminium chloride, basic (CAS 1327-41-9)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the similar behaviour regarding toxicokinetics, data from the different soluble Aluminium salts are used in read-across approach to assess the toxicological properties of Aluminium sulphate. Hence, the data from the other soluble Aluminium salts are considered as a starting point in the hazard assessment of Aluminium sulphate taking into account the differences in bioavailability using available toxicokinetic information.

4. DATA MATRIX
See attached justification
Reason / purpose:
read-across source
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:
Based on the read-across approach, the target substance 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. typhimuriumstrains TA1535, TA1537, TA98 and TA100 andE.colistrain 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 inS. typhimuriumstrains TA1535, TA1537, TA98 and TA100, andE.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 at 100, 300 and 600μg/mL for a 3 h exposure time with a 27 h harvest time in the absence and presence of S9-fraction. The experiment with S9 -miw was repeated because precipitate interfered with the scoring for the cytokinesis-block proliferation index; the concentrations of 100, 300, 400, 500 and 600 µg/mL were tested. In this experiment, no precipitate was observed and the concentrations 300, 500 and 600 µg/mL were selected for scoring of micronuclei. In the second cytogenetic assay, test material was tested at 100, 300 and 600μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. For this experiment, Cytochalasine B (5μg/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 600 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 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 333μg/mL 24 h treatment, without S9-mix: 1, 3, 10, 33, 100, 250, 333 and 500 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, the relative suspension growth was 77% at the highest test substance concentration of 725μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 76 % at the highest test substance concentration of 725μg/mL compared to the relative suspension growth of the solvent control. After 24 h of treatment without S9-mix, the relative suspension growth was 14 % at the test substance concentration of 725μg/mL compared to the relative suspension growth of the solvent control.Test material precipitated in the exposure medium at concentrations of 363μg/mL and above in all experimental conditions.

In the first mutagenicity test, the relative total growth was 84% and 103% at 333μg/mL compared to the total growth of the solvent controls at 3 h treatment without and with 8% S9-mix, respectively. Precipitation was observed at the highest dose tested in both experimental conditions, i.e. 333 µg/mL.In the second mutagenicity test, the relative total growth was 28% at 500 µg/mL and 103% at 333μg/mL compared to the total growth of the solvent controls for the 24 h treatment without S9 -mix and for the 3 h treatment with 12% S9-mix, respectively. Precipitation was recorded from 250 µg/mL and at 333 µg/mLfor the 24 h treatment without S9-mix and for the 3 h treatment with S9-mix, respectively. The t

est 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, Klimisch Grade 1 in vivo micronucleus studies on aluminium hydroxide and dialuminium chloride pentahydroxide (Locron P) have 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 or dialuminum cjloride pentahydroxide to aluminium chloride 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 chloride basic.

A total of four in vitro studies and 3 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 or equivocal 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 inadequately 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 were not observed in the highly reliable GLP studies performed in laboratories, which are skilled in making such observations. In these studies, exposure levels and durations 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.