Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14/07/2006 to 21/07/2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP compliance and International Guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ACQUAPOL C1

IUPAC Name:

ACQUAPOL C1

Test material form:

other: liquid

Details on test material:

Identification: ACQUAPOL C1
Physical state: Liquid

Method

Target gene:

Organism-test: Salmonella typhimurium

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

microsomal fraction of rat liver induced with Aroclor 1254

Test concentrations with justification for top dose:

648; 1080; 1800; 3000; 5000 µg/plate

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

For the test minimal glucose agar and top agar were used and prepared following the procedures described by Maron and Ames (1983). In each tubewith 3 mL, of "top agar" and previously placed oin a dry bath at 45 °C was added 0.1 mL of a fresh bacterial culture grown overnight and 0.1 mL of test substance. For tests with metabolic activation 0.5 mL/plate of S9 prepared by "Molecular Toxycology Incorporated" (Annapolis, EUA) was added. The concentration of protein in S9 fraction employed in this assay was 40.8 mg/mL..

Previously of the test, the genotypes of the strains were checked for ensure the genetic characteristic original of bacteria.

Controls
Concurrent positive and negative controls were included in each assay. Negative controls consisting of vehicle (deionized water, 100 µL/plate) without the test substance were included in each assay to obtain the number of revertants colonies per plate to compare with the number of reverants observed in the presence of the test substance. Positive controls should ensure both strain responsiveness and efficacy of the metabolic activation system. The chemicals employed in the test are listed in table 1.

Concentration:
A range finding assay using TA100 was performed to select concentrationsfor the definitve test at the following concentrations: 8, 40, 200, 1000, 5000 µg/plate. No concentration was found to be toxic for bacteria growing Salmonella typhimurium. Therefore the definitive test with the strains TA98 and TA100 was performed at the following concentration: 648; 1080; 1800;3000; 5000 µg/plate. all plating was done in triplicate in the absence and presence of metabolic activation. The negative controls were done in triplicate and the positive controls were done in two replicates.

Results were presented as number of revertant colonies per plate and concentration and by the mutation rate (MR), which corresponds to the rate between number of revertants induced by the test substance and number of revertants iobserved in the negative control.

Evaluation criteria:

A result is considerred to be active when the average number of revertent colonies in test plates is equal to or higher than double that observed in negative control plates (MR>2) for the strains TA100 and TA98. To confirm the positive rsult the analysis of variance of the data set should indicate significant result of pANOVA < 5 % and a clear dose-related increase in the number of revertants should be observed. The analysis of variance indicate the probability of the number of revertants observed in the different concentrations be increased (mutagenicity) or decreased (toxicity).

The acceptance criteria of the assay are: a) presence of background lawn in the test plates; b) spontaneous revertant colonies of the negative control are in the range reported in the literature and estabilished in the laboratory by historical control values; c) positive controls show mutagenic activity in all tested strains.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment with ACQUAPOL C1 at any concentration level in the presence or absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acnowleddged border of biological significance. Mutation rates were lower than 2 for the tested strains in the absence of metabolic activation.
The analysis of variance of the data set indicated no significant difference (pANOVA >0.05) for the strain TA98 in the absence of metabolic activation. In the assays with strain TA100 the analysis of variance of the data set indicated significant difference (pANOVA >0.05). In these tests we observed an increase in the number of revertants with increasing concentrations, but no biological significance, since the folding was no observed in the number of revertants in the test concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion the test substance is not mutagenic in the condition of the test.

Executive summary:

The reverse mutation assay (Ames Test) was carried out with the product ACQUAPOL C1 in order to study the possible mutagenic effect of that substance on the strains TA98 and TA 100 of Salmonella Typhimurium in system with and without metabolic activation (microsomal fraction of rat liver induced with Aroclor 1254). The definitive test was performed at the foillowing concentrations: 648, 1080,1800,3000,5000 µg/plate. Mutation frequencies after 72 hours of incubation of Salmonella Typhimurium strains were lower than 2. Statistical analysis presented no significant results (pANOVA>0.05) for strain TA98 and was significant for strain TA100. Under the conditions of this study, ACQUAPOL C1 has presented no mutagenic effect on Salmonella typhimurium strains TA98 and TA100 both with and without metabolic activation. The substance is not mutagenic in the condition of the test.