Tall oil, maleated

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:

Study Initiation:October 8, 2013 Experimental Start:October 9, 2013 Experimental Completion: November 1, 2013 Study Completion:February 24, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, maleated

EC Number:

268-859-6

EC Name:

Tall oil, maleated

Cas Number:

68152-93-2

Molecular formula:

UVCB

IUPAC Name:

3,7-dimethyl-14,16-dioxo-19-(propan-2-yl)-15-oxapentacyclo[10.5.2.0²,¹¹.0³,⁸.0¹³,¹⁷]nonadeca-13(17),18-diene-7-carboxylic acid; 8-(7-hexyl-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)octanoic acid

Test material form:

liquid

Details on test material:

Lot No.: HD0258QH13

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

Remarks:

histidine auxotrophs

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other:

Remarks:

tryptophan-deficient

Metabolic activation:

with and without

Metabolic activation system:

Rat S9

Test concentrations with justification for top dose:

Based on preliminary testing, the main study doses were designed to reach the test item’s solubility limit of approximately 2 mg/plate in the test system.

For the plate incorporation test, EnvaMulTM 600 was tested at a maximum exposure concentration of 2.0 mg/plate, in the presence and absence of S9.

In the pre-incubation test, EnvaMulTM 600 was tested at a maximum concentration of 2.0 mg/plate for most tester strains with the exception of TA1537 without S9 where the maximum analyzable concentration was 0.25 mg/plate, due to test item toxicity.

Vehicle / solvent:

DMSO
In preliminary testing, EnvaMulTM 600 was determined to be insoluble in water. DMSO was determined to be a suitable solvent as EnvaMulTM 600 was soluble at 20 mg/mL.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Method of application: plate incorporation experiment and pre-incubation test.

Evaluation criteria:

Negative result (no evidence of genotoxicity) is concluded if there is no substantial increase in the number of colonies per plate; i.e. the results do not exceed the upper 98 percentile limit of the historical solvent/negative control range. A negative result indicates that the test item is non-mutagenic in S. typhimurium or E. coli.
Positive result will be considered positive when there is a significant increase in the number of colonies per plate in comparison to the concurrent negative control and a concentration-related increase over the exposure range tested. A positive result indicates that the test item induces point mutations in S. typhimurium or E. coli.
Equivocal result: If no definite judgment can be made to fit the above criteria, even after repeated experiments, then the result will be described as equivocal. An equivocal result indicates that a definitive result cannot be made by performing the bacterial reverse mutation assay under the conditions described in this study plan.

Statistics:

The numbers of revertant colonies per plate are represented as colony counts in Table 1 for the plate incorporation test and in Table 2 for the pre-incubation test. The tables include the individual plate counts, the means and standard deviations (S.D.). These means and standard deviations were plotted against the dose levels in Figures 1 to 10 for the plate incorporation test and Figures 11 to 20 for the preincubation test. All colony counts were compared to the historical data from this laboratory. The historical ranges in Appendix V are defined by the minimum and maximum mean colony counts. The 95% confidence intervals are also reported.
Statistical analysis was applied to the numbers suspected to be abnormally high or to have a dose-related increase in revertant counts. The colony counts were transformed (square root) to normalize the data prior to using the one-sided Dunnett’s test (Mahon,G.A.T., et al., 1989).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The pre-incubation test resuts confirmed the plate incorporation test results as negative.

Applicant's summary and conclusion

Conclusions:

Tall Oil, Maleated (EnvaMulTM 600) was not mutagenic to S. typhimurium strains, TA98, TA100, TA1535, TA1537, and E. coli strain, WP2 uvrA, under the conditions of the test.

Executive summary:

Tall Oil, Maleated (EnvaMulTM 600) was evaluated for its potential to induce point mutations in Salmonella typhimurium strains, TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA. The experimental design followed the “OECD Guideline for Testing of Chemicals - 471, Bacterial Reverse Mutation Test” (OECD, 1997).

In the plate incorporation experiment, Tall Oil, Maleated (EnvaMulTM 600) denoted as EnvaMulTM 600, was tested at a highest concentration of 2.0 mg/plate in the presence and absence of S9. At this level, slight precipitate was visible with a microscope. Precipitate was not observed for any lower concentrations. A clear pattern of toxicity was not observed for all conditions when compared to the concurrent negative controls. For all conditions, a normal background lawn was observed but for some conditions, a greater than 30% reduction in colony counts from the concurrent negative controls was observed. Thus, the test item was evaluated at the limit of solubility in the test system (OECD, 1997). Finally, for all strains and conditions, all 5 concentrations were analyzable for mutagenicity (OECD, 1997). For all conditions, the test item did not produce any statistically significant increases (p>0.01) in colony counts over the concurrent negative controls and a dose-response was not observed. A more sensitive pre-incubation test was designed to confirm the negative results of the plate incorporation test.

In the pre-incubation test, EnvaMulTM 600 was tested at a maximum concentration of 2.0 mg/plate for all tester strains; however, due to extreme toxicity at 0.5, 1.0, and 2.0 mg/plate, for TA1537 without S9, the maximum concentration evaluated for this strain was 0.25 mg/plate. Slight and moderate precipitate were observed under the microscope at 1.0 and 2.0 mg/plate without S9, respectively. Slight precipitate was observed with a microscope at 2.0 mg/plate with S9. Test item precipitate was not observed for all lower concentrations. For some conditions, a reduction of the background lawn and/or the number of colony counts was observed for at least the highest concentration. Only for TA1537 without S9 was toxicity clearly the limiting factor in the selection of concentrations tested. In this case, test item toxicity reduced the maximum analyzable concentration from 2.0 mg/plate to 0.25 mg/plate. Therefore, EnvaMulTM 600 was tested at the limit of solubility in the test system for most conditions with the exception of TA1537 without S9 where the test item was tested at the limit of toxicity (OECD, 1997). For all strains and conditions, at least 5 concentrations were analyzable for mutagenicity (OECD, 1997). For all conditions, the test item did not produce any statistically significant increases (p>0.01) in colony counts over the concurrent negative control and a dose-response was not observed. Therefore, the pre-incubation test was concluded as negative and confirmed the results of the plate incorporation test.

Thus, it was concluded that Tall Oil, Maleated (EnvaMulTM 600) was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain, WP2 uvrA, under the test conditions.