Rosin, reaction products with acrylic acid

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:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Rosin, reaction products with acrylic acid

EC Number:

280-192-2

EC Name:

Rosin, reaction products with acrylic acid

Cas Number:

83137-13-7

Molecular formula:

N.A. - UVCB substance

IUPAC Name:

Rosin, reaction products with acrylic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot No. 646729
- Expiration date of the lot/batch: 19 November 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: The test article formed a clear solution in DMSO at a concentration of approximately 299 mg/mL in the solubility test conducted at BioReliance.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
To achieve a solution, the most concentrated dilution was vortexed for 3 to 4 minutes in each assay. Test article dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test article dissolved in DMSO

Method

Target gene:

Histidine operon (S. typhimurium) and tryptophan (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The Salmonella typhimurium tester strains were from Dr. Bruce Ames, University of California, Berkeley. The tester strains may also be obtained from Molecular Toxicology Inc. (Moltox)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The E. coli tester strain was from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland (United Kingdom). The tester strains may also be obtained from Molecular Toxicology Inc. (Moltox)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay (with and without metabolic activation): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg/plate
Mutagenicity assay (with and without metabolic activation): 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate (top dose based on the results of the preliminary toxicity assay)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test article and compatibility with the target cells.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation
- Cell density at seeding (if applicable): 1.8 to 3.9 x 10^8 cells per plate

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

Test conditions were based on the protocols in OECD Guideline 471 (Bacterial Reverse Mutation Assay).

Evaluation criteria:

The following criteria must be met for the mutagenicity assay to be considered valid:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.

Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. As appropriate, colonies were enumerated either by hand or by machine.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.

The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.

A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Statistics:

Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:

Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay with and without metabolic activation (S9-mix). 

Executive summary:

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the presence and absence of metabolic activation (S9-mix). The test method was based on OECD 471 and Ames et al. (1973) and was performed in compliance with GLP. The test article was dissolved in DMSO. A dose range-finding test was performed with concentration up to 5000 μg/mL in the absence and presence of metabolic activation. Based on the results of the dose range finding test, the test article was dosed at concentrations of 15.0, 50.0, 150, 500, 1500, and 5000 μg per plate. Each culture was run in triplicate. The following procedure was carried out for each strain. One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50.0 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45°C. When plating the positive controls, the test article aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48-72 hours at 37°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using codes ranging from normal (1 or no code) to absent (6; complete lack of any microcolony lawn over greater than or equal to 90% of the plate). As appropriate, colonies were enumerated either by hand or by machine. No substantial increases in the revertant colony numbers of any of the tester strains were observed following treatment with the test article at any dose level with and without metabolic activation. Vehicle and positive controls responded as expected. Based on the results of the study, the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay with and without metabolic activation (S9-mix).