Tall oil, potassium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Tall oil, potassium salt

EC Number:

271-968-1

EC Name:

Tall oil, potassium salt

Cas Number:

68647-71-2

Molecular formula:

Not applicable. It is not possible to assign a specific molecular structure to a UVCB substance.

IUPAC Name:

Tall oil, potassium salt

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

Details on test material:

- Appearance: amber coloured paste
- Storage conditions: room temperature in the dark

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.

Additional strain / cell type characteristics:

other: Histidine mutation G46; rfa; uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.

Additional strain / cell type characteristics:

other: Histidine mutation D3052; rfa; uvrB; pkM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

The actual batch of the strain was tested for ampicillin resistance, UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances. The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.

Additional strain / cell type characteristics:

other: Histidine mutation G46; rfa; uvrB; pkM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (derived from rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

Experiment 1
0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of metabolic activation for all strains tested.

Experiment 2
0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of metabolic activation in strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was not sufficiently soluble in water.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: The results of the first experiment were verified by a second, independent experiment. Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted and for the positive controls. For the vehicle control groups, six-fold repetitions were run.

PERFORMANCE OF THE TEST
- Conditions of cultivation: One day prior to the test, a small amount from each of the frozen bacterial cultures was transferred to nutrient broth. The liquid cultures were incubated in a shaker overnight at 37 °C and then used for the exposure.
- Exposure technique: For each sample the following solutions were combined: 0.1 mL of the overnight culture of the bacteria, 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation), 0.1 mL of the appropriate test or reference material solution and 2 mL of top agar
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
- Colony counting: The plates with less than about 50 revertant colonies, with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
- Determination of the toxicity: The bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded: a reduced bacterial background lawn (mottled instead of homogeneous), microcolonies of bacteria instead of a homogeneous background lawn, no background lawn or clearly reduced numbers of revertant colonies.

Evaluation criteria:

1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response.
5. Statistical analysis of data as determined by UKEMS.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA1535, TA1537,TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was non mutagenic to the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA both in the presence and absence of metabolic activation.

Executive summary:

The mutagenic activity of the test material was investigated in a reverse mutation test in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA were exposed to the test material in DMSO using the direct plate incorporation method both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The bacteria were also exposed to vehicle and appropriate positive controls. The concentrations tested were 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. An independent, repeat experiment was conducted with pre-incubation method at 0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results. Under the conditions of this study, the test substance was non mutagenic to the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA both in the presence and absence of metabolic activation (Wisher, 2019).