Dialuminium chloride pentahydroxide

Genetic toxicity: in vitro

Administrative data

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:

22-April-2010 to 10-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.

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: particulate/powder

Details on test material:

- Name of test material (as cited in study report): 202240/A
- Physical state: solid, white crystalline powder
- Expiration date of the lot/batch: 13 December 2010
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable

Method

Target gene:

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

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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: 100, 333, 1000, 3330 and 5000 µg/plate

Experiment 2:
TA1535
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
TA1537
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
TA98
Without S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Experiment 3
TA1535
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
TA1537
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
TA98
Without S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
TA100 and WP2uvrA
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test substance was dissolved in Milli-Q 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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene in DMSO

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. Three 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

Key result

Species / strain:

S. typhimurium 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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:
- Toxicity was observed at dose levels of 3330 μg/plate and above in the absence of S9-mix in tester strain TA100. Toxicity was observed at the dose level of 5000 μg/plate in the presence of S9-mix in tester strain TA100 and in the absence and presence of S9-mix in tester strain WP2uvrA. of

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 and with S9: 3330 µg/plate and above
TA1537: without and with S9: 1000 µg/plate and above
TA98: without S9: 333 µg/plate and above and with S9: 3330 µg/plate and above
TA100: without and with S9: 3330 µg/plate and above
WP2uvrA: without and with S9: 5000 µg/plate

Any other information on results incl. tables

Since in the tester strains TA1535 and TA98 in the absence of S9-mix and in TA1537 in the absence and presence of 5% (v/v) S9-mix in the first experiment too many dose levels with toxicity were tested, this part of the experiment was repeated (experiment 2).

Applicant's summary and conclusion

Conclusions:

Under the test conditions, 202240/A is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.

Executive summary:

A bacterial reverse mutation assay (Ames Test) was performed according to the OECD guideline No. 471 and in compliance with GLP. The test material 202240/A was tested in Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in Escherichia coli (WP2uvrA). The test was performed in three independent experiments in the presence and absence of S9-mix (phenobarbital and ß-naphthoflavone-induced rat liver S9). The dose range finding study was reported as part of Experiment 1.

- Dose range finding:

TA100 and WP2uvrA : up to 5000 µg/plate with and without S9 mix (5%)

- Experiment 1:

TA1535, TA1537, TA98: up to 5000 µg/plate with and without S9 mix (5%)

- Experiment 2:

TA1535: up to 3330 µg/plate without S9 mix

TA1537: up to 1000 µg/plate with and without S9 mix (3.6%)

TA98: up to 1000 µg/plate without

- Experiment 3:

TA1535: up to 3330 µg/plate without S9 mix ; up to 5000 µg/plate with S9 mix (10%)

TA1537: up to 1000 µg/plate with and without S9 mix (10%)

TA98: up to 1000 µg/plate without S9 mix ; up to 5000 µg/plate with S9 mix (10%)

TA100 and WP2uvrA: up to 5000 µg/plate with and without S9 mix (10%).

202240/A did not precipitate on the plates at the highest dose levels.

Toxicity was observed in all tester strains in all experiments. Since in the strains TA1535 and TA98 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix too many dose levels with toxicity were tested, this part of the experiment was repeated in mutation experiment 2.

202240/A did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in two independently repeated experiments.

In this study, 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, 202240/A is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.