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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLPs and according to OECD guideline 471, the European Commission Annex V Test Method 813/814, ICH Guidelines CPMP/ICH/141/95 Step 4 and the USA EPA Pesticide Assessment Guidelines Sub-division F, Series 84-2.
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
Remarks:
Type of genotoxicity: gene mutation
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 GLPs and according to OECD guideline 471, the European Commission Annex V Test Method 813/814, ICH Guidelines CPMP/ICH/141/95 Step 4 and the USA EPA Pesticide Assessment Guidelines Sub-division F, Series 84-2.
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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His (-), Trp (-)
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 tester strains were received from B.N. Ames, Department of Biochemistry, University of California, Berkeley, CA, USA and stored in liquid nitrogen until use.
Additional strain / cell type characteristics:
other: rfa and uvr B mutations
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The E. coli strain was obtained from the National Collection of Industrial Bacteria, Aberdeen, UK
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes from the livers of male Fischer 344 rats that had been injected with Aroclor 1254.
Test concentrations with justification for top dose:
Toxicity and Mutation Assays:
17, 50, 167, 500, 1667 and 5000 µg per plate.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-Aminoanthracene was dissolved in dimethylsulphoxide and used with all tester strains in the presence of S9 activation at dose concentrations of 2 µg/plate (TA 1535 and TA 1537), 0.5 µg/plate (TA 98 and TA 100), or 20 µg/plate (E. coli WP2 uvr A).
Positive control substance:
sodium azide
Remarks:
Sodium azide was dissolved in sterile, ultra-pure water and was used with tester strains TA 100 and TA 1535 in the absence of S9 activation at a dose concentration of 1 µg/plate.
Positive control substance:
9-aminoacridine
Remarks:
9-Aminoacridine was dissolved in dimethylsulphoxide and was used with tester strain TA 1537 in the absence of S9 activation at a dose concentration of 80 µg/plate.
Positive control substance:
2-nitrofluorene
Remarks:
2-Nitrofluorene was dissolved in dimethylsulphoxide and was used with tester strain TA 98 in the absence of S9 activation at a dose concentration of 1 µg/plate.
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine was dissolved in dimethylsulphoxide and was used with tester strain E. coli WP2 uvr A in the absence of S9 activation at a dose concentration of 2 µg/plate.
Details on test system and experimental conditions:
Toxicity Test
-A toxicity test using strain TA 100 only was performed in the presence and absence of S9 activation to determine final dose concentrations.

Mutation Tests
-Two independent mutation tests were conducted using all tester strains; the first employed the direct plate method and the second employed the pre-incubation 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 Medium E with 2% glucose.

Preparation of bacteria:
-Samples of each strain were grown by culturing in 25 g Oxoid Nutrient Broth No 2 for 16 h at 37 °C.

Preparation of assay plates:
-For S. typhimurium strains, sterile 1.0 mM L-histidine.HCl/1.0 mM biotin solution was added, in 5 mL per 100 mL of soft agar. For E. coli WP2 uvr A, 1.0 mL of 1.35 mM L-tryptophan was added per 100 mL agar. The agars were thoroughly mixed prior to use and maintained in a water bath at 45 °C.

The direct plate method:
-To 2 mL of soft agar, the following were added: 0.5 mL of S9 mix or 0.05 M phosphate buffer, followed by 0.1 mL of bacteria, with the solvent or test solution (0.05 mL) added last. The tube contents were mixed and poured onto minimal medium plates containing 25 mL of 1.5% purified agar in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and incubated at 37 °C for 2 or 3 days.

The pre-incubation method:
-S9 mix or 0.05 M phosphate buffer were dispensed into sterile tubes with 0.1 mL of bacteria per tube and the solvent or test solution (0.05 mL per tube). The tube tops were then screwed on tightly and placed in a shaking incubator at 37 °C for 20 min upon which time the contents were combined with 2 mL of soft agar. The tube contents were then mixed and poured onto minimal medium plates containing 25 mL of 1.5% purified agar in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and incubated at 37 °C for 2 or 3 days.

After incubation, the colonies were counted using a Cardinal Colony Counter (Perceptive Instruments, UK) and the plates were examined for precipitates and microscopically for microcolony growth.

Study dates:
-Study Initiation: April 17, 2001
-Experimental Start Date: May 9, 2001
-Experimental Completion Date: June 29, 2001
-Study Completion Date: December 13, 2001
Evaluation criteria:
A test was considered acceptable if, for each strain:
-the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
-at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2 uvr A 1-60.
- for at least 2 of the positive control plates there were 2X (1.5X for TA 100) the mean vehicle control mutant numbers per plate.
-no toxicity or contamination was observed in at least 4 dose levels.
-in cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
-S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli, at least a doubling (or 1.5X for TA 100) of the mean concurrent vehicle control values at some concentration of the test item.
-a dose related response
-a reproducible effect in independent tests.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

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 in line with historical data on those substances.
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:
other: no; tested up to and including precipitating concentrations. The two highest concentrations tested caused some precipitation but lawns were assumed to be normal.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no; tested up to and including precipitating concentrations. The two highest concentrations tested caused some precipitation but lawns were assumed to be normal.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
-Toxicity test:
No toxicity to the bacteria was observed, but precipitation of the test material occurred at 1667 and 5000 µg per plate in both the presence and the absence of S9 mix. At 5000 µg per plate, the background lawn of microcolonies was obscured by the excessive precipitation. This made accurate assessment of toxicity difficult but it was assumed that the lawns were normal and that there was no toxicity.

-Mutation tests:
No toxicity to the bacteria was observed but precipitation of the test material occurred at 1667 and 5000 µg per plate in both the presence and the absence of S9 mix. At 5000 µg per plate, the background lawn of microcolonies was obscured by the excessive precipitation making accurate assessment of toxicity difficult but it was assumed that the lawns were normal and that there was no toxicity.

-Quality control:
All strains were sensitive to crystal violet; TA 98 and TA 100 strains were resistant to ampicillin. The strains were also tested for sensitivity to UV light emitted over a period of 5-10s at 254 nm and an increased sensitivity to UV light was demonstrated.

-Vehicle control:
The vehicle control values were within the normal ranges obtained at the testing facility.

-Positive control groups:
The results obtained were within the normal ranges obtained at the testing facility and were in the normal ranges expected for each bacterial strain and activation condition.

-Test rejection:
All parts of the assays were acceptable according to the study criteria except those assays involving TA 1537 and WP2 uvr A in the first mutation test, which were contaminated. These two parts were repeated successfully.
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

Based on an absence of genotoxic/mutagenic effects, Resin acids and rosin acids, hydrogenated, Me esters 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.

It is concluded that, under the conditions of this test, this rosin showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation.
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, Me esters in ethanol at concentrations of 17, 50, 167, 500, 1667 and 5000 µg per plate in the presence and absence of mammalian metabolic activation using both the plate incorporation and pre-incubation methods. At the two highest concentrations tested, precipitation of the test substance occurred and to some extent obscured the background lawns of the microcolonies. However, the background lawns were assumed to be normal, without evidence of toxicity. There was no evidence of an increase in induced mutant colonies over background with the test substance at any concentration tested, while the positive controls induced the appropriate responses in the corresponding strains.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance:
-Test substance (as cited in report): Rosin, partially hydrogenated methyl ester
-Lot number: CMP-8039
-Physical properties: amber liquid
-Test substance receipt: June 13, 2000
-Storage: dark and at ambient temperature
-Purity: 100%
-Expiration: June 15, 2005

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 tester strains were received from B.N. Ames, Department of Biochemistry, University of California, Berkeley, CA, USA and stored in liquid nitrogen until use.
Additional strain / cell type characteristics:
other: rfa and uvr B mutations
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The E. coli strain was obtained from the National Collection of Industrial Bacteria, Aberdeen, UK
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes from the livers of male Fischer 344 rats that had been injected with Aroclor 1254.
Test concentrations with justification for top dose:
Toxicity and Mutation Assays:
17, 50, 167, 500, 1667 and 5000 µg per plate.
Vehicle / solvent:
Ethanol
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-Aminoanthracene was dissolved in dimethylsulphoxide and used with all tester strains in the presence of S9 activation at dose concentrations of 2 µg/plate (TA 1535 and TA 1537), 0.5 µg/plate (TA 98 and TA 100), or 20 µg/plate (E. coli WP2 uvr A).
Positive control substance:
sodium azide
Remarks:
Sodium azide was dissolved in sterile, ultra-pure water and was used with tester strains TA 100 and TA 1535 in the absence of S9 activation at a dose concentration of 1 µg/plate.
Positive control substance:
9-aminoacridine
Remarks:
9-Aminoacridine was dissolved in dimethylsulphoxide and was used with tester strain TA 1537 in the absence of S9 activation at a dose concentration of 80 µg/plate.
Positive control substance:
2-nitrofluorene
Remarks:
2-Nitrofluorene was dissolved in dimethylsulphoxide and was used with tester strain TA 98 in the absence of S9 activation at a dose concentration of 1 µg/plate.
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine was dissolved in dimethylsulphoxide and was used with tester strain E. coli WP2 uvr A in the absence of S9 activation at a dose concentration of 2 µg/plate.
Details on test system and experimental conditions:
Toxicity Test
-A toxicity test using strain TA 100 only was performed in the presence and absence of S9 activation to determine final dose concentrations.

Mutation Tests
-Two independent mutation tests were conducted using all tester strains; the first employed the direct plate method and the second employed the pre-incubation 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 Medium E with 2% glucose.

Preparation of bacteria:
-Samples of each strain were grown by culturing in 25 g Oxoid Nutrient Broth No 2 for 16 h at 37 °C.

Preparation of assay plates:
-For S. typhimurium strains, sterile 1.0 mM L-histidine.HCl/1.0 mM biotin solution was added, in 5 mL per 100 mL of soft agar. For E. coli WP2 uvr A, 1.0 mL of 1.35 mM L-tryptophan was added per 100 mL agar. The agars were thoroughly mixed prior to use and maintained in a water bath at 45 °C.

The direct plate method:
-To 2 mL of soft agar, the following were added: 0.5 mL of S9 mix or 0.05 M phosphate buffer, followed by 0.1 mL of bacteria, with the solvent or test solution (0.05 mL) added last. The tube contents were mixed and poured onto minimal medium plates containing 25 mL of 1.5% purified agar in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and incubated at 37 °C for 2 or 3 days.

The pre-incubation method:
-S9 mix or 0.05 M phosphate buffer were dispensed into sterile tubes with 0.1 mL of bacteria per tube and the solvent or test solution (0.05 mL per tube). The tube tops were then screwed on tightly and placed in a shaking incubator at 37 °C for 20 min upon which time the contents were combined with 2 mL of soft agar. The tube contents were then mixed and poured onto minimal medium plates containing 25 mL of 1.5% purified agar in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and incubated at 37 °C for 2 or 3 days.

After incubation, the colonies were counted using a Cardinal Colony Counter (Perceptive Instruments, UK) and the plates were examined for precipitates and microscopically for microcolony growth.

Study dates:
-Study Initiation: April 17, 2001
-Experimental Start Date: May 9, 2001
-Experimental Completion Date: June 29, 2001
-Study Completion Date: December 13, 2001
Evaluation criteria:
A test was considered acceptable if, for each strain:
-the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
-at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2 uvr A 1-60.
- for at least 2 of the positive control plates there were 2X (1.5X for TA 100) the mean vehicle control mutant numbers per plate.
-no toxicity or contamination was observed in at least 4 dose levels.
-in cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
-S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli, at least a doubling (or 1.5X for TA 100) of the mean concurrent vehicle control values at some concentration of the test item.
-a dose related response
-a reproducible effect in independent tests.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

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 in line with historical data on 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:
other: no; tested up to and including precipitating concentrations. The two highest concentrations tested caused some precipitation but lawns were assumed to be normal.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no; tested up to and including precipitating concentrations. The two highest concentrations tested caused some precipitation but lawns were assumed to be normal.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
-Toxicity test:
No toxicity to the bacteria was observed, but precipitation of the test material occurred at 1667 and 5000 µg per plate in both the presence and the absence of S9 mix. At 5000 µg per plate, the background lawn of microcolonies was obscured by the excessive precipitation. This made accurate assessment of toxicity difficult but it was assumed that the lawns were normal and that there was no toxicity.

-Mutation tests:
No toxicity to the bacteria was observed but precipitation of the test material occurred at 1667 and 5000 µg per plate in both the presence and the absence of S9 mix. At 5000 µg per plate, the background lawn of microcolonies was obscured by the excessive precipitation making accurate assessment of toxicity difficult but it was assumed that the lawns were normal and that there was no toxicity.

-Quality control:
All strains were sensitive to crystal violet; TA 98 and TA 100 strains were resistant to ampicillin. The strains were also tested for sensitivity to UV light emitted over a period of 5-10s at 254 nm and an increased sensitivity to UV light was demonstrated.

-Vehicle control:
The vehicle control values were within the normal ranges obtained at the testing facility.

-Positive control groups:
The results obtained were within the normal ranges obtained at the testing facility and were in the normal ranges expected for each bacterial strain and activation condition.

-Test rejection:
All parts of the assays were acceptable according to the study criteria except those assays involving TA 1537 and WP2 uvr A in the first mutation test, which were contaminated. These two parts were repeated successfully.
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

Based on an absence of genotoxic/mutagenic effects, Resin acids and rosin acids, hydrogenated, Me esters 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.

It is concluded that, under the conditions of this test, this rosin showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation.
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, Me esters in ethanol at concentrations of 17, 50, 167, 500, 1667 and 5000 µg per plate in the presence and absence of mammalian metabolic activation using both the plate incorporation and pre-incubation methods. At the two highest concentrations tested, precipitation of the test substance occurred and to some extent obscured the background lawns of the microcolonies. However, the background lawns were assumed to be normal, without evidence of toxicity. There was no evidence of an increase in induced mutant colonies over background with the test substance at any concentration tested, while the positive controls induced the appropriate responses in the corresponding strains.