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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity in bacteria (OECD 471): negative with and without metabolic activation Chromosome aberration in vitro (OECD 473) on analogue: negative with and without metabolic activation Gene mutation in mammalian cells (OECD 476) on analogue: negative with and without metabolic activation
Link to relevant study records
Reference
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:
30-04-2014 to 26-05-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to the guidelines, under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine
E-coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 from rats induced with Phenobarbitone/ß-Naphthoflavone
Test concentrations with justification for top dose:
plate incorporation (test 1): 1.5, 5. 15, 50, 150, 500, 1500 and 5000 ug/plate
pre-incubation assay (tet 2): 50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA100, TA1535 and WP2uvrA without metabolic activation
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA100, TA1535, TA 1537 and WP2uvrA with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA98 without metabolic acivation
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA98 with metabolic activation
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA1537 without metabolic activation
Positive control substance:
9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st test in agar (plate incorporation); 2nd test preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth rate bacterial bachground lawn

COLONY COUNT:
Domino colony counter

Evaluation criteria:
A test item will be considered mutagenic (positive) in the test system if one or more of these criteria are met
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
Statistics:
Not specified according to UKEMS
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, but tested up to precipitating concentrations
Remarks:
film at 5000 ug/plate
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, but tested up to precipitating concentrations
Remarks:
film at 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.
Executive summary:

The test substance was tested in an Ames test at concentrations upto the preciptation level (5000 ug/plate). In a plate incorporation and a pre-incubation assay (both performed in triplicate), the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in bacteria

Resin acids and rosin acids, fumarated, compds. with triethanolamine was tested in an Ames test at concentrations up to the

precipitation level (5000 ug/plate) (Harlan 2014). In a plate incorporation and a pre-incubation assay (both performed in triplicate), the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.

In a bacterial reverse point mutation Ames test with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2uvrA conducted according to GLP and OECD 471, Rosin, fumarated was negative in increasing the number of revertant colonies when tested at 17, 50, 167, 500, 1667, and 5000 ug/plate with and without metabolic activation system from liver of rats treated with Aroclor 1254 (Stevenson, 2001).

In a bacterial reverse point mutation Ames test with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 conducted according to GLP and OECD 471, Rosin, fumarated was negative in increasing the number of revertant colonies when tested at 50, 158, 500, 1580, 500 ug/mL3 with and without metabolic activation (May, 1991).

 

Mammalian chromosomal aberrations in vitro       

In a mammalian cell gene mutation assay conducted in accordance with GLP and OECD 473, Chinese hamster ovary cells cultured in vitro were exposed to Rosin, fumarated in DMSO (Murie, 2002). The following concentrations were tested: 10, 20, and 40 ug/mL, +S9, for Test 1; 39, 78, and 156 ug/mL, -S9, for Test 1; 30, 40 and 50 ug/mL, +S9, for Test 2; 80, 95, and 110 ug/mL, -S9, for Test 2 at the 24-hour harvest; 40, 80, and 120 ug/mL, -S9, for Test 2 at 48-hour harvest. Rosin, fumarated did not show clastogenic effects under the conditions of the study, both in the presence and absence of activation system.

Mammalian gene-cell mutation

A study conducted with Rosin, fumarated evaluated the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (Flanders, 2010). The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to ten dose levels using a 4hour exposure group in the presence of metabolic activation (1% S9) and a 24hour exposure group in the absence of metabolic activation. The dose range for the first experiment was 2.5 to 80 µg/ml in the absence of metabolic activation, and 5 to 160 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 5 to 80 µg/ml in the absence of metabolic activation, and 5 to 120 µg/ml in the presence of metabolic activation. The maximum dose level used in the mutagenicity test was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

The overall data on the mutagenic potential of resin acids and rosin acids, fumarated, compds. with triethanolamine do not show

mutagenic or clastogenic activity in bacterial and/or mammalian cells in vitro in tests performed with either the test substance or the component Rosin, fumarated.

Justification for selection of genetic toxicity endpoint
study according to the guidelines under GLP with the test substance, which confirms the findings found on components of the UVCB test substance

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

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