Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride

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:

30 January 2017 - 27 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride

Molecular formula:

not applicable

IUPAC Name:

Reaction products of Resin acids and Rosin acids, sodium salts and barium chloride

Test material form:

solid: particulate/powder

Details on test material:

Name as cited in the report: Barium salts of resin acids and rosin acids
Appearance: light tan powder
Storage conditions: at room temperature desiccated

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:

Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 1: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: ethanol is accepted and approved by authorities and international guidelines.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191 2.5 µg/plate in DMSO for TA1537 (for experiment 1 only)

Remarks:

without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

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 ± 4 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted (one direct plate assay and one pre-incubation assay)

NUMBER OF CELLS EVALUATED: 10E8 per plate

PREPARATION OF PLATES:
First experiment (direct plate assay): The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture of one of the tester strains, 0.1 mL of a dilution of the test item in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark.
Second experiment (pre-incubation assay): The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture of one of the tester strains, 0.05 mL of a dilution of the test item in ethanol. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark.

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or 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 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test substance is considered positive (mutagenic) if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 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 follow up 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:

no cytotoxicity

Vehicle 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium, other: TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 512 μg/plate (for TA1537 without S9) and at 5000 μg/plate (for other strains)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 164 μg/plate (without S9) and from 5000 μg/plate (with S9)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium, other: TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 512 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding test: at the start and at the end of the incubation period at concentrations of 1600 and 5000 μg/plate (+/- S9-mix).
Experiment 1: at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 and 5000 μg/plate at the end of the incubation period (+/- S9-mix).
Experiment 2: at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 and 5000 μg/plate at the end of the incubation period (+/- S9-mix).
Experiment 3: at the start and at the end of the incubation period at concentrations of 1600 and 5000 μg/plate, except in tester strain TA1535, where precipitation was also observed at 512 μg/plate at the end of the incubation period (+/- S9-mix).

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

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.

OTHER:
- In tester strain TA1537, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed in the absence and presence of S9-mix. Since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.
- In strain TA100, fluctuations in the mean number of revertant colonies above the laboratory historical control data range were observed in the absence and presence of S9-mix. However since the increases were not two-fold (a maximum of 1.2-fold was reached), these increases were not considered to be relevant.
- In the second and third experiment: Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants at this dose level could not be determined.

Remarks on result:

other: Direct plate assay

Any other information on results incl. tables

The following acceptability criteria were met, therefore the study was considered valid:

a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) did exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.

b) The selected dose range extended to 5 mg/plate.

c) No plates were lost throughout the study.

The 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, except the response for WP2uvrA in the absence of S9-mix in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline and GLP principles, Barium salts of resin acids and rosin acids was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline and GLP principles. Three experiments were conducted: one direct plate assay, one pre-incubation assay and one additional pre-incubation assay. The latter was conducted to verify the response observed in the second experiment. In the first experiment, no relevant responses were seen for toxicity or mutagenicity. In the second mutation experiment, the test item induced an up to 2.9- and 6.4-fold increase in the number of colonies compared to the solvent control in the tester strains WP2uvrA and TA1535 in the absence and presence of S9-mix, respectively. The increase in tester strain WP2uvrA was within the laboratory historical control data range. The increase in tester strain TA1535 was outside the laboratory historical control data range in only one out of three plates. Both increases were not seen in the first experiment or in the additional experiment. Therefore these increases are considered to be not biologically relevant. All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the 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.

Based on the results of this study it is concluded that Aluminium tribenzoate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.