Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro assays were performed to evaluate the genotoxicity of the test substance (OECD 471, 473 and 476, under GLP conditions). The test item or an analogue substance, respectively, did not induce mutations in bacterial or mammalian cells and did not induce chromosomal aberrations in CHL cells. Therefore, the substance is considered as non-mutagenic under the conditions of these tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

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:

S9 mix from phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

20 μg - 5 000 μg/plate (SPT)
8 μg - 2 000 μg/plate (PIT)

Vehicle / solvent:

Due to the limited solubility of the test item in water, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD),

Remarks:

without S9 mix

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours

NUMBER OF REPLICATIONS: in triplicate

NUMBER OF CELLS EVALUATED: all revertants / colonies counted

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
was recorded for all test groups both with and without S9 mix in all experiments.

Evaluation criteria:

The test item is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test item is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was found from 2500 μg/plate onward with and without S9 mix.
A bacteriotoxic effect was observed depending on the strain and test conditions from about 1 000 μg/plate onward

Conclusions:

Interpretation of results:
negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital and β-naphthoflavone induced rats

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL
with S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL

2nd Experiment
without S9 mix (24-hour exposure period)
0; 0.16; 0.31; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00 μg/mL
with S9 mix (4-hour exposure period)
0; 4.69; 9.38; 18.75; 37.50; 75.00; 150.00 μg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 6-7 days
- Selection time (if incubation with a selection agent): one week

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
pH
Osmolarity
solubility
cell morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within the historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest concentrations evaluated

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality: no effect
- Precipitation: In culture medium test substance precipitation occurred by the end of treatment at concentrations of 39.1 μg/mL and above in the absence and presence of S9 mix

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Conclusions:

Interpretation of results:
negative

Under the experimental conditions of this study, the test substance was not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see the attached justification.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro assays were performed to evaluate the genotoxicity of the test substance.

At first, the material 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 (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test item was applied at concentrations of 20 μg - 5000 μg/plate (standard plate test) and 8 μg - 2000 μg/plate (pre-incubation test) in the presence and absence of a metabolic activation system (S9 mix). Precipitation of the test item was found from 2500 μg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

 

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation). The test item was applied at concentration of 0.78 - 200 µg/ml and cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. In this study, in the 1st and 2nd experiment, the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, the test substance is not mutagenic in the HPRT locus

assay.

 

read across to CAS 110 -30 -5

The test item was not examined for its potential to induce chromosomal aberrations in mammalian cells. Reliable experimental data on a read across substance are available. The analogue is a 2-fold amidated C16 fatty acid complex and belongs to the Fatty Acid Reaction Products with Amines sub-group of the Fatty Nitrogen Derived Amides Categories of the HPV program. The actual test item is an UVCB which consists mainly of 6-fold aminated C17 fatty acids. Solubility and bioavailability of the test material is dominated by the length of the fatty acid chain which is comparable to the analogue substance. The difference in the number of amino groups (grade of amidation) plays a minor role due to the availability of one free amino group only (potential Schiff`base formation and subsequent DNA-interaction).

The in vitro chromosome aberration test of the structural analogue was conducted by using a female Chinese hamster lung cell line (CHL/IU). The substance, dissolved in DMSO, was applied at concentrations up to 500 μg/ml (highest technically feasible concentration) for 6h or 24h in the presence and absence of a metabolic activation system (S9 mix). Cytotoxicity was not observed. The appearance frequency of cells with structural or numerical chromosome aberrations was lower than 5 % at any doses. The substance is therefore not considered to cause chromosomal aberrations.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EU) 2020/217.