Amides, from C8-10-fatty acids and tetraethylenepentamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across based on structural analogue, CAS 68555 -22 -6

3 standard in vitro studies were carried out according to OECD test guidelines. Each were determined to be negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

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.

Justification for type of information:

Refer to read across justification document attached in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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:

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.

Justification for type of information:

Refer to read across justification document attached in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Updated and adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch number: BBD02611V1-1.STEP

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:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

All tester strains without S9: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA100, TA1535 and TA1537 with S9: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA98 and WO2uvrA with S9: 15, 50, 150, 500 and 1500 µg/plate

Top dose determined by preliminary study that showed precipitate at doses >1500µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.

For the test substance to be evaluated positive, it must cause a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, reaction products of C8-10-fatty acids and tetraethylenepentamine cyclics, branched and linear did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, reaction products of C8-10-fatty acids and tetraethylenepentamine cyclics, branched and linear, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Precipitate was observed beginning at 3333 µg per plate. Toxicity was observed beginning at concentrations from 100 to 3333 µg per plate. Based upon these results, the maximum doses tested in the mutagenicity assay were 1500 µg per plate with all tester strains in the absence of S9 activation and tester strains TA100, TA1535 and TA1537 in the presence of S9 activation and 5000 µg per plate with tester strains TA98 and WP2uvrA in the presence of S9 activation.

In the mutagenicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate with all tester strains in the absence of S9 activation and tester strains TA100, TA1535 and TA1537 in the presence of S9 activation and 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with tester strains TA98 and WP2uvrA in the presence of S9 activation. Precipitate was observed at 5000 µg per plate. Toxicity was observed beginning at 150, 500 or 1500 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate reaction products of C8-10-fatty acids and tetraethylenepentamine cyclics, branched and linear was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine 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 Fatty acids C16-18, C18 unsat reaction products with tetraethylenepentamine is not mutagenic in theSalmonella typhimuriumreverse mutation assay.

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.

 

Tall oil, reaction products with tetraethylenepentamine 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.

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.

Therefore, it is conclude that Tall oil, reaction products with tetraethylenepentamine (Amidoamine/Imidazoline) is not clastogenic in human lymphocytes.

 

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. 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,Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline)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,Tall oil, reaction products with tetraethylene-pentamine (Amidoamine/Imidazoline)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 therefore are considered to be not genotoxic.

 

Short description of key information:

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

Endpoint Conclusion: No adverse effect observed (negative)

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 not genotoxic.