Fatty acids, tall-oil, reaction products with diethylenetriamine

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:

Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with diethylenetriamine

EC Number:

263-160-2

EC Name:

Fatty acids, tall-oil, reaction products with diethylenetriamine

Cas Number:

61790-69-0

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

Fatty acids, tall-oil, reaction products with diethylenetriamine

Test material form:

other: solid, waxy, white to yellowish

Specific details on test material used for the study:

- Source and lot/batch No.of test material: 00173191511
- Expiration date of the lot/batch: 2018-12-18
- Storage condition of test material: room temperature

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

standard plate test: up to 5000µg/plate
pre-incubation assay: up to 5000µg/plate (e.coli), up to 1000µg/plate (salmonella strains)
Doses limited by bacteriotoxicity to Salmonella strains starting at 100µg/plate or above (depending on tester strain) and precipitation (observed from 1000µg/plate onward)

Vehicle / solvent:

Acetone
Reason for choice of vehicle: The test substance is hardly soluble in water, but dissolves well in acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (all strains w/ S9); N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, TA100 w/out S9); 4-nitro-o-phenylenediamine (TA98 w/out S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20min (pre-incubation assay)
- Exposure duration: 48-72h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in.the number of revertant colonies within the range of the historical positive control data or.above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Test substance precipitation was found from about 1000 μg/plate onward with and without S9 mix.

Any other information on results incl. tables

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	100 – 5000	1000 – 5000	1000 – 5000	333 – 5000	-
	With	1000 – 5000	1000 – 5000	1000 – 5000	333 – 5000	-
2nd-PIT	Without	333 – 1000	333 – 1000	100 – 1000	333 – 1000	-
	With	333 – 1000	333 – 1000	333 – 1000	333 – 1000	-

Reduced background growth was observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	-
	With	-
2nd-PIT	Without	333 – 1000	100 – 1000	100 – 1000	1000	-
	With	333 – 1000	333 – 1000	100 – 1000	333 – 1000	-

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or marginally above the range of the historical negative control data for each tester strain.

In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range or above of the historical positive control data.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose Range: 33 µg - 5000 µg/plate (SPT), 3.3 µg - 5000 µg/plate (PIT)

Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

Solubility: Precipitation of the test substance was found from about 1000 µg/plate onward with and without S9 mix.

Toxicity: A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 µg/plate onward.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.