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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471, EU Method B.13/14, OPPTS 870.5100, OPPTS 870.5265, and GLPs.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471, EU Method B.13/14, OPPTS 870.5100, OPPTS 870.5265, and GLPs.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test substance was non-toxic and did not cause any visible reduction in the bacterial background lawn at any dose concentration.

Mutagenicity Test:
A white, opaque film was observed at 5000 µg/plate, but this did not prevent the scoring of revertant colonies. The test substance did not cause any visible reduction in the bacterial background lawn at any dose concentration with or without metabolic activation.

Negative controls:
Results were considered to be acceptable and within the range of the historical control.

Positive controls:
The positive control chemicals induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Resin acids and Rosin acids, hydrogenated, potassium salts showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation.

Based on an absence of genotoxic/mutagenic effects, Resin acids and Rosin acids, hydrogenated, potassium salts is not classifiable for Germ Cell Mutagenicity according to UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Resin acids and Rosin acids, hydrogenated, potassium salts is not classified in Annex I of Directive 67/548/EEC.
Executive summary:

This data is being read across from the source study that tested Resin and rosin acids, hydrogenated, potassium salt based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A of E. coli were exposed to Resin acids and Rosin acids, hydrogenated, potassium salts in sterile distilled water at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the direct plate incorporation method. At the highest concentration tested, a white opaque film was observed but this did not affect the scoring of revertant colonies. There was no evidence of an increase in induced mutant colonies over background with the test substance at any concentration tested, while the positive, negative and vehicle controls induced the appropriate responses in the corresponding strains.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Test material (as cited in report): Resin acids and Rosin acids, hydrogenated, potassium salts
-Batch/Lot number: X29363-079
-Date received: 2004-08-25
-Physical state: pale beige/white solid
-Storage conditions: room temperature in the dark

Method

Target gene:
His (-), Trp (-)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The S. typhimurium strains were received from the University of California, Berkeley, California, USA on 1995-08-04 and stored on culture discs at -196 °C in liquid nitrogen until use.
Additional strain / cell type characteristics:
other: rfa and uvrB mutations
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The Escherichia coli strain WP2 uvr A was obtained from the British Industrial Biological Research Association on 1987-08-17 and stored at -196 °C in liquid nitrogen until use.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from livers of male Sprague-Dawley rats previously treated with 3 daily oral doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg/day). Before use, each batch of S9 was assayed for its metabolic activity. The S9 was stored at -196°C.
Test concentrations with justification for top dose:
Preliminary toxicity study:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation test:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
Sterile distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was dissolved in sterile distilled water and used with tester strains TA 100 (3 µg/plate), TA 1535 (5 µg/plate) and WP2 uvr A (2 µg/plate) without metabolic activation.
Positive control substance:
9-aminoacridine
Remarks:
9-Aminoacridine (9AA) was dissolved in sterile distilled water and used with tester strain TA 1537 (80 µg/plate) without metabolic activation
Positive control substance:
other: 4-Nitroquinoline-1-oxide (4NQO):
Remarks:
4-Nitroquinoline-1-oxide (4NQO) was dissolved in sterile distilled water and used with tester strain TA 98 (0.2 µg/plate) without metabolic activation.
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
2-Aminoanthracene (2AA) was dissolved in sterile distilled water and used with tester strains TA 100 (1 µg/plate), TA 1535 (2 µg/plate), TA 1537 (2 µg/plate), and WP2 uvr A (10 µg/plate) with metabolic activation.
Positive control substance:
benzo(a)pyrene
Remarks:
Benzo(a)pyrene (BP) was dissolved in sterile distilled water and used with tester strains TA 98 (5 µg/plate) with metabolic activation.
Details on test system and experimental conditions:
-Preliminary Toxicity Test:
The test was conducted using the plate incorporation method. A preliminary toxicity test using tester strains TA 100 and WP2 uvr A only was performed in the presence and absence of S9 activation to determine final dose concentrations. The doses used were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

-Mutation Tests:
Two independent mutation tests were conducted using all tester strains employing the direct plate method. Each experiment was performed in triplicate with each bacterial strain and dose level in both the presence and the absence of S9 activation.

-Agar Plates:
The agar plates were Vogel-Bonner Minimal Agar (30 mL/plate).

-Preparation of bacteria:
Sub-cultures were prepared in nutrient broth (Oxoid Limited; lot number 323907 11/08) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

-Preparation of Test and Control Materials:
Solubility checks determined that at 50 mg/mL, the test material was fully soluble in sterile distilled water so this was selected as the vehicle. The test material was weighed and half-log dilutions were prepared in sterile distilled water by mixing on a vortex mixer for 30 minutes at 40 ºC on the day of the each experiment.

-Controls:
Vehicle controls (sterile distilled water), negative controls (concurrent untreated plates) and positive controls were used in parallel with the test material.

-Preparation of assay plates:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar; 0.1 mL of the test material formulation, vehicle or positive control; and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Evaluation criteria:
The test was considered valid if the following criteria were met:
-All tester strain cultures exhibited a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-The appropriate characteristics for each tester strain were confirmed (e.g. rfa cell-wall mutation and pKM 101 plasmid R-factor etc.)
-All tester strain cultures were in the approximate range of 1 to 9.9 X 10^9 bacteria per mL.
-Each mean positive control value was at least twice the respective vehicle control value for each strain.
-There were a minimum of four non-toxic test substance dose levels.
-There was no evidence of excessive contamination.

Where these criteria were met, a significant mutagenic response was recorded if:
The test substance induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression; statistical analyses were not required due to the absence of an increase in the number of revertant colonies at any dose level. Results for the positive control materials were consistent with historical data for those substances.

Results and discussion

Test resultsopen allclose all
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test substance was non-toxic and did not cause any visible reduction in the bacterial background lawn at any dose concentration.

Mutagenicity Test:
A white, opaque film was observed at 5000 µg/plate, but this did not prevent the scoring of revertant colonies. The test substance did not cause any visible reduction in the bacterial background lawn at any dose concentration with or without metabolic activation.

Negative controls:
Results were considered to be acceptable and within the range of the historical control.

Positive controls:
The positive control chemicals induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Resin acids and Rosin acids, hydrogenated, potassium salts showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation.

Based on an absence of genotoxic/mutagenic effects, Resin acids and Rosin acids, hydrogenated, potassium salts is not classifiable for Germ Cell Mutagenicity according to UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Resin acids and Rosin acids, hydrogenated, potassium salts is not classified in Annex I of Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A of E. coli were exposed to Resin acids and Rosin acids, hydrogenated, potassium salts in sterile distilled water at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the direct plate incorporation method. At the highest concentration tested, a white opaque film was observed but this did not affect the scoring of revertant colonies. There was no evidence of an increase in induced mutant colonies over background with the test substance at any concentration tested, while the positive, negative and vehicle controls induced the appropriate responses in the corresponding strains.