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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine (FA+TEPA) was found not to be mutagenic in a bacterial mutagenicity study (Ames test), induced no chromosomal aberrations in a study in human lymphocytes, and was not mutagenic in a mammalian mutagenicity study in mouse lymphoma cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
25 Aug 2000 - 14 Nov 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 471 and in compliance with GLP.
Justification for type of information:
Also see category read across justification document attached to chapter 13
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Read across is done from (source) Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine, CAS 68991-84-4 (Tallow-TEPA) to (target) Fatty acids C18 unsat, reaction products with tetraethylenepentamine, CAS 1226892-45-0 (Tall-TEPA)

These amidoamine/imidazolines are made from fatty acid and polyethyleneamines. The manufacturing process is a one-step process with formation of amide and imidazoline structures. To promote imidazoline formation from the amide, the reaction mixture is heated to temperatures above 180ºC. The resulting product therefore is a mixture of the amide structure of the fatty acid and the polyethyleneamine and its imidazoline.
The production of Tallow-TEPA is the same as Tall-TEPA, except for a small difference in chain lengths distribution between tallow derived alkyl chains and tall-oil derived alkyl chains in the fatty acid source used in starting materials. As a consequence, the resulting structures are chemically and structurally completely comparable with only difference in the alkyl chain distribution reflecting the starting material.

Tall oil is basically consisting of C18-unsaturated and some C16 chains, and tallow consists of a mix of C16 (about 25%), C18 (up to 20%) and C18-unsatuarted (up to 50%), and a small percentage other chain lengths.
As consequence, these products are for 50% completely identical, whereas for the other 25% the Tallow-TEPA has a slightly shorter alkyl chain compared to the Tall-TEPA. For all other aspects, the substances are identical. As both have the same chemical structure they both have identical functional groups, show the same chemical reactivity and have very comparable physicochemical properties. Consequently, they share the same mechanism of action. This is similarly applicable to all constituents of these UVCBs.
 
Within a specific structure, the variability of the alkyl chain length is considered to have a possible modifying activity, which is related to modification of the physiological properties of the molecule with increase or shortening of the apolar alkyl chain part. This is suspected to influence aspects related to bioavailability, but not aspects of chemical reactivity and route of metabolism, aspects that influence specific mechanisms of toxicity such as sensitisation and genotoxicity and are more related to the hydrophilic part. As the differences in chain lengths are only very minimal as all substances basically contain C16 and C18 alkyl chains, it seems justified from a toxicological point of view to consider the fatty acid part as similar for all AAI substances.

Specifically for skin genotoxicity, the molecular structure with functional groups are essential with respect to the identification of possible mutagenic properties. In that respect, Tallow-TEPA and Tall-TEPA are identical.
Related to the (small) difference in average alkyl chain-lengths between the two substances, the Tallow-TEPA with on average a slightly shorter chain-length than the Tall-TEPA, is expected to be slightly better bioavailable (slightly smaller molecule also leading to more moles per unit of weight, slightly higher solubility, slightly lower Kow, and slightly higher vp) and is expected to have (a slightly) higher bioavilability compared to Tall-TEPA.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction from Moltox (Molecular Toxicology, INC, Annapolis, MD 21401,USA); liver of rats treated with Aroclor 1254 (500 mg/kg) i.p.
Test concentrations with justification for top dose:
Without S9-mix:
- 12.5,25,50, 100 and 200 µg/plate: for all tester strains in the first experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for all tester strains in the second experiment,
- 1.5625, 3.125, 6.25, 12.5 and 25 µg/plate: for all tester strains in the third experiment.
With S9-mix:
- 12.5, 25, 50, 100 and 200 µg/plate: for all tester strains in both experiments, except for the TA 1537 strain in the second experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for the TA 1537 strain in the second experiment.
Vehicle / solvent:
DMSO
Positive controls:
yes
Positive control substance:
other: Sodium-azide 1 µg/plate (TA100, TA1535); 2-Nitrofluorene 0.5µg/plate (TA98); 9-Aminoacridine 50 µg/plate (TA1537); Mitomycin C 0.5µg/plate (TA102).
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Anthramine: 2 µg/plate (TA1535, TA1537, TA98, TA100), 10 µg/plate (TA102)
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation: (preliminary toxicity test, all experiments without 89 mix, first experiment with 89 mix); preincubation ((second experiment with 89 mix)

DURATION
- Exposure duration: 48 to 72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted on each strain with and without S9 mix, and a third experiment on TA98, TA100 and TA102 strains, without S9 mix.

DETERMINATION OF CYTOTOXICITY
Six dose-levels (one plate/dose-level) were tested in the TA 98, TAl00 and TA 102 strains, with and without 89 mix.
Based on decrease in the number ofrevertant colonies and/or a thinning ofthe bacterial lawn.

OTHER EXAMINATIONS:
In each experiment, the following controls were included using triplicate plates:
. vehicle controls: each bacterial tester strain treated with the vehicle,
. positive controls: each bacterial tester strain treated with appropriate reference mutagens.
The sterility of the 89 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproducible two-fold increase in the number ofrevertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation ofthe data obtained.
Statistics:
Not indicated.
Species / strain:
other: All Salmonella strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With S9 ≥ 500µg/plate; without S9 ≥ 100µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:
- 12.5,25,50, 100 and 200 µg/plate: for all tester strains in the first experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for all tester strains in the second experiment,
- 1.5625, 3.125, 6.25, 12.5 and 25 µg/plate: for all tester strains in the third experiment.

In the first experiment, a slight to marked toxicity was noted in the tester strains generally at dose-levels ≥ 100 µg/plate.
In the second experiment, a slight toxicity was induced in the TA 1535 and TA 1537 strains at 100 µg/plate. In the three remaining tester strains, the toxicity induced was more important than the first experiment: a slight to strong toxicity, depending on the dose-levels and the tester strains. Due to the variability in toxicity and in order to avoid too toxic dose-levels, a third experiment was performed with these three tester strains at lower dose-levels.
In this third experiment, except for a slight thinning of the bacterial lawn noted in the TA 100 strain at dose-levels ≥ 12.5 µg/plate, no toxicity was observed.

In the three experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.

Experiments with S9 mix:
- 12.5, 25, 50, 100 and 200 µg/plate: for all tester strains in both experiments, except for the TA 1537 strain in the second experiment,
- 6.25, 12.5,25,50 and 100 µg/plate: for the TA 1537 strain in the second experiment.

In the first experiment, except for a moderate toxicity noted in the TA 1537 and TA 98 strains at 200 µg/plate, no noteworthy toxicity was induced.
In the second experiment (preincubation method), a slight to moderate toxicity was noted in all tester strains, generally at dose-levels ≥ 100 µg/plate.

In both experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The negative and strain-specific positive control values indicate that the test conditions were adequate and that the metabolic activation system functioned properly. Under the experimental conditions of this study, the test substance does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The objective of this study was to evaluate the potential of the test substance BASE 136 (batch No. AB771) to induce reverse mutation inSalmonella typhimurium.

 

Methods:

A preliminary toxicity test was performed to define the dose-levels of BASE 136 to be used for the mutagenicity study. The test substance was then tested in two independent experiments with and three independent experiments without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

 

All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

 

Five strains of bacteriaSalmonella typhimurium:TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test substance BASE 136 was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

Without S9 mix:

- 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

- 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

- 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

- 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

 

With S9mix:

- 2 µg/plate of2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

- 10 µg/plate of2-Anthramine (2AM): TA 102 strain.

 

Results

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix:

- 12.5,25,50, 100 and 200 µg/plate: for all tester strains in the first experiment,

- 6.25, 12.5,25,50 and 100 µg/plate: for all tester strains in the second experiment,

- 1.5625, 3.125, 6.25, 12.5 and 25 µg/plate: for all tester strains in the third experiment.

 

A slight to marked toxicity was induced in the tester strains, depending on the dose-levels.

 

In the three experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.

 

Experiments with S9 mix:

- 12.5, 25, 50, 100 and 200 µg/plate: for all tester strains in both experiments, except for the TA 1537 strain in the second experiment,

- 6.25, 12.5,25,50 and 100 µg/plate: for the TA 1537 strain in the second experiment.

 

A slight to moderate toxicity was induced, depending on the tester strain, the dose-levels and the experimental conditions.

 

In both experiments, no noteworthy increase in the number of revertants was observed in all the tested strains.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

Conclusion

Under our experimental conditions, the test substance BASE 136 (batch No. AB771) does not show mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

These results on Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine are fully valid for Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline). Their respective fatty acid moieties are only marginally differing in chain lengths distribution, and it is considered that their reaction products with TEPA are principally the same when evaluating for genotoxicty.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-jul-2009 to 03-oct-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 10, 20, 30, 50, 70 and 100 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 20, 30, 100 and 333 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 10, 20, 30, 50, 70 and 100 µg/mL
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 10, 30 and 70 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 30, 70 and 100 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 60 and 80 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 20 and 30 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 30 and 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9-mix

Migrated to IUCLID6: : in Hank's Balanced Salt Solution: 0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix

Migrated to IUCLID6: : in Hank's Balanced Salt Solution: 10 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Species / strain:
lymphocytes: human peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 333 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 50 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 100 and 20 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr, respectively and at dose levels of 50 µg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

It was noted that Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay, in the absence of S9-mix at the 24 h exposure time and in the presence of S9-mix in the second cytogenetic assay. This may indicate that Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) has the potential to inhibit mitotic processes.

No effects of Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline)was studied for its effect on the number ofon the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix) in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch S000925 of Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was a clear slightly viscous amber liquid. Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was dissolved in ethanol.

 

In the first cytogenetic assay, Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was tested up to 70 and 100 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively.

Appropriate toxicity was reached at these dose levels.

 

In the second cytogenetic assay, Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was tested up to 80 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 30 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was tested up to 100 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

 

It was noted that Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay, in the absence of S9-mix at the 24 h exposure time and in the presence of S9-mix in the second cytogenetic assay. This may indicate that Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) has the potential to inhibit mitotic processes.

 

No effects of Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

 

Finally, it is concluded that this test is valid and that Tall oil, reaction products with tetraethylenepentamine (Amidoamine/Imidazoline) is not clastogenic in human lymphocytes under the experimental conditions described in this report. Tall oil, reaction products with tetraethylenepentamine (Amidoamine/Imidazoline) may have the potential to disturb mitotic processes and to induce numerical chromosome aberrations.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-aug-2009 to 07-oct-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
- Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100, 333 and 500 µg/mL
Without S9-mix, 24 hours treatment: 2.7, 8, 27, 80, 270 and 400 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 6, 10, 16 and 20 µg/mL
With S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 30, 50, 60 and 70 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1, 3, 5, 6, 7, 8, 9 and 10 µg/mL
With S9-mix, 3 hours treatment: 10, 30, 50, 60, 70, 80 and 90 μg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: 7.5 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

RANGE-FINDING/SCREENING STUDIES:
-The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controls) + 126 in a dose-dependent manner; or
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 270 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 10 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 100 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 8 µg/mL in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 73 and 91% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 82 and 87% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Remarks on result:
other: strain/cell type: L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) is not mutagenic in the TK mutation test system under the experimental conditions described in the report.
Executive summary:

Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline)was evaluated for its possible induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix(rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study was performed under GLP and according to the most recent OECD and EU guidelines.

 

Batch S000925 of Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/ Imidazoline) was a clear slightly viscous amber liquid. The test substance was dissolved in ethanol.

 

In the first experiment, Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/ Imidazoline) was tested up to concentrations of 20 and 70 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was tested up to cytotoxic levels of 73 and 93% in the absence and presence of S9-mix, respectively.

 

In the second experiment, Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/ Imidazoline) was tested up to concentrations of 10 and 90 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) was tested up to cytotoxic levels of 82% and in the absence of S9-mix and up to 87% in the presence of S9-mix.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Except the response of one of the solvent-treated control cultures in the absence of S9-mix (first experiment), this mutation frequency was above the limit of the historical control data range. However since all observed mutation frequencies of the solvent control substances were within the acceptability criteria of this assay, the validity of the test was considered to be not affected.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 12-fold for MMS in the absence of S9-mix, and by 11- and 7.9-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/ Imidazoline) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

 

It is concluded that Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For each endpoint, bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity, a recent guideline and GLP compliant study is available.

 

In vitro gene mutation study in bacteria:

Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine (FA+TEPA) was tested in the Salmonella typhimurium reverse mutation assay with five histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced with Aroclor 1254). The study followed the most recent OECD and EU protocols and was performed under GLP.

There was no significant or dose-related increase in the number of revertant colonies in any of the applied strains, both with and without S9-mix. This was confirmed in an independently repeated experiment.

It is concluded that FA+TEPA is not mutagenic in the Salmonella typhimurium reverse mutation assay.

 

In vitro cytogenicity study in mammalian cells:

Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) (FA+TEPA) was studied for its effect on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix), in two independent experiments. The study was performed under GLP and according to the most recent OECD and EU guidelines.

FA+TEPA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

It was noted that FA+TEPA increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay, in the absence of S9-mix at the 24 h exposure time and in the presence of S9-mix in the second cytogenetic assay. This may indicate that FA+TEPA has the potential to inhibit mitotic processes. No effects of FA+TEPA on the number of cells with endo-reduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

Therefore, it is concluded that FA+TEPA is not clastogenic in human lymphocytes.

 

In vitro mutagenicity study in mammalian cells:

Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline) (FA+TEPA) was evaluated for its possible induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix. The study was performed under GLP and according to the most recent OECD and EU guidelines.

In both the presence and absence of S9-mix, FA+TEPA did not induce a significant increase in the mutation frequency in the first experiments. This result was confirmed in a repeat experiment with modifications in the duration of treatment time (without S9-mix) or S9 concentration (with S9-mix). Therefore, FA+TEPA is not mutagenic in the TK mutation test.

 

Also other AAI (DETA, and PolyEA based AAI, including a substance consisting of only Amidoamine without imidazoline) have similarly been tested, with the same results.

AAI substances in general are therefore considered to be not genotoxic.

Justification for classification or non-classification

Tall oil, reaction products with tetraethylenepentamine is not mutagenic in the Salmonella typhimurium reverse mutation assay (based on test with Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine), is not clastogenic in human lymphocytes, and not mutagenic in the TK mutation test with L5178Y mouse lymphoma cells.

It can therefore be concluded that Tall oil, reaction products with tetraethylenepentamine is not genotoxic.