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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
11 days (02 August 2010 to 13 August 2010)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline 471 and EU Method B13/B14 with no deviations that affected the results of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Resin acids and rosin acids, calcium salt, CAS 9007-13-0
- Supplier: Flint Group
- Supplier code: GZ2005Z
- Physical state: Solid
- Color: Amber
- Analytical purity: 100%
- Lot/batch No.: not provided
- Expiration date of the lot/batch: February 2011
- Stability under test conditions: Not provided
- Storage condition of test material: room temperature and protected from light

Method

Target gene:
His (-) and 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):
Master and bacterial banks were prepared from lyophilized bacterial discs. Stock plates were stored at -80 °C +/-10.
Additional strain / cell type characteristics:
other: See table in "Any other information on materials and methods including tables" section.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
Master and bacterial banks were prepared from lyophilized bacterial discs. Stock plates were stored at -80 °C +/-10.
Additional strain / cell type characteristics:
other: See table in "Any other information on materials and methods including tables" section.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
5000, 1667, 556, 185 and 62 μg/plate
Vehicle / solvent:
corn oil
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
5 µg/plate for S. typhimurium TA98 in the absence of S9; DMSO as solvent
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2.5 µg/plate for S. typhimurium TA100 and TA1535 in the absence of S9; H2O as solvent
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
30 µg/plate for S. typhimurium TA1537 in the absence of S9; DMSO as solvent
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.4 µg/plate for E. coli WP2 (pKM101) in the absence of S9; DMSO as solvent
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1.5 µg/plate, 2.5 µg/plate, 30 µg/plate, 10 µg/plate, and 5 µg/plate for Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 (pKM 101), respectively in the presence of S9; DMSO as solvent
Details on test system and experimental conditions:
Salmonella strains TA 98, TA 100, TA 1535 and TA 1537 and E. coli strain WP2 were tested with 62, 185, 556, 1667 and 5000 µg/plate of the test material suspended in corn oil. The test was initially run using the direct incorporation method. The confirmatory test was run using the pre-incubation method.

INITIAL TEST (direct incorporation method): Cultures of bacteria were incubated overnight with nutrient broth up to an absorbance (660nm) of approximately 1.2-1.4 OD. This OD indicates that bacteria are growing in the late exponential or early stationary phase of growth (approximately 10^9 bacteria/mL). Plates were prepared with minimal agar medium. Medium was mixed and preheated to about 45 ºC, and then poured into the plate and cooled to room temperature. Each bacterial strain was tested in triplicate in the presence and absence of the metabolic system (S9). The bacterial suspension, the test item and phosphate buffered saline (-S9) or metabolic activation system mix (+S9) were mixed and tempered at about 37 ºC. The suspension was mixed with top agar and poured over minimal agar medium plate. The top agar was allowed to solidify at room temperature before final incubation. Plates were incubated at about 37 ºC for about 72 hours. A negative solvent control and appropriate positive controls for each strain and experimental condition were included in the experiment.


CONFIRMATION TEST (pre-incubation method): An independent confirmation test was performed with the test item according to the preincubation procedure. After the bacterial suspension, the test item and phosphate buffered saline (-S9) or metabolic activation system mix (+S9) were mixed, and the mixture was incubated at about 37 ºC for about 20 minutes. Thereafter, the study was performed in the same way as the direct incorporation method.

STERILITY TEST: Sterility of the test item and the S9 mix were tested by adding the highest concentration of the test material and a sample of S9 mix to top agar preheated to 45 ºC and poured over minimal agar medium plates. Plates were incubated for 72 hours at 37 ºC and then observed for the presence or absence of colonies. The presence of bacterial growth would indicate the presence of microbiological contamination.

SOLUBILITY TEST: Solubility was assessed as precipitate formation in the final mixture under the actual test conditions used. Observation of precipitation by the naked eye indicated insolubility.

CYTOTOXICITY TEST: A dose-dependent reduction in number of colonies compared to the negative control might indicate cytotoxicity.

PREPARATION OF METABOLIC ACTIVATION SYSTEM: The S9 was prepared using 10% v/v S9 fraction, 8.01 mM MgCl2, 33 mM KCl, 4.64 mM Glucose-6-phosphate, 4.01 mM NaDPH, and 0.2 M phosphate buffer.

DATA PRESENTATION: The number of colonies per plate was counted with an automatic colony counter. Data were presented as the number of colonies present per plate (mean ± standard deviation). The ratio R was calculated as follows: R = Number of revertant colonies in the presence of the test substance/Number of revertant colonies in the absence of the test item.
Evaluation criteria:
Criteria used for determining a positive result were a dose-response in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Positive results from the bacterial reverse mutation test indicate that the test substance induces point mutations or frame-shifts in the genome of the tested bacterial strains. Negative results from the test indicate that under the test conditions, the test substance was neither mutagenic nor pro-mutagenic in the tested experimental system.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
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
Additional information on results:
The controls of the test were in concordance with the expected results. Sterility test showed no contamination during the study. No cytotoxic effect was observed. All positive controls showed valid ratios (R) above 2.5. Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. No concentration of the test substance showed a biological significant increase (R ≥ 2.5) in the number of revertant either with or without S9 metabolic activation. No dose response was observed for any of the tested 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 (migrated information):
negative with metabolic activation
negative without metabolic activation

At a maximum concentration of 5000 µg/plate, Resin acids and rosin acids, calcium zinc salts did not induce mutation in Salmonella typhimurium strains TA 98, TA 100, TA 1535 or TA 1537, or in E. coli strain WP2 when tested in the bacterial reverse mutation test (Ames assay) in the presence and absence of a metabolic activation system. The test material was not cytotoxic and no experiment with the test substance showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation. No dose response increase in colony formation was observed for any of the tested bacterial strains.

Based on an absence of genotoxic/mutagenic effects in this study, Gum Rosin is not classifiable for Germ Cell Mutagenicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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 of E. coli were exposed to Resin acids and rosin acids, calcium zinc salts in corn oil at concentrations of 62-5000 µg/plate in the presence and absence of a mammalian metabolic activation system using both the plate incorporation method and the pre-incubation method. No cytotoxicity was observed at any dose level up to the limit concentration of 5000 µg/plate. No experiment with the test substance showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation. No dose response was observed for any of the tested bacterial strains. All vehicle and positive controls induced the appropriate responses in the corresponding strains. Based on the test conditions used in this study, Resin acids and rosin acids, calcium zinc salts was found to be neither mutagenic nor pro-mutagenic.