Aluminum chloride, basic

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid - liquid: aqueous solution

Details on test material:

- Physical state: Clear yellow liquid
Liquid
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiration date of the lot/batch: 18 December 2010

- Other:
General information: Forms polymeric ions and Al(OH)3 when diluted with water, depending on pH
Density: 1.363 kg/L at 20°C
pH: 1.0 ± 0.5% (20°C)

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Controls 2

Positive control substance:

9-aminoacridine

Remarks:

without S9: 60 µg/plate in water for TA1537

Controls 3

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98

Controls 4

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: 650 µg/plate in DMSO for TA100

Controls 5

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Controls 6

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Applicant's summary and conclusion

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.