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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Oleyl tripropylenetetramine was not mutagenic in a bacterial mutagenicity study (Ames test) and not clastogenic and/or aneugenic to cultured human lymphocytes. The similar product Tallow (C16-C18, C18 unsat.) tripropylenetetramine was not mutagenic in a mammalian mutagenicity study in mouse lymphoma cells. All studies were performed under GLP according to current guidelines.

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:
experimental study
Adequacy of study:
key study
Study period:
16-jun-2009 to 29-jun-2009
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Substance as described in Chapter 1.1
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537, TA98 and TA100:
Without and with S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
Experiment 2:
TA1535
Without and with S9-mix: 0.3, 1, 3, 10, 20 and 33 µg/plate
TA1537, TA98 and TA100
Without and with S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
WP2uvrA
Without and with S9-mix: 1, 3, 10, 33, 100 and 200 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
: ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9 Migrated to IUCLID6: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: 2-aminoanthracene in DMSO for all strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

OTHER:
- Precipitation of the test substance on the plate
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
b) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) The increase in the mean number of revertant colonies follows the concentration of test substance (dose-response relationship).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 33 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 µg/plate and above in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 33 µg/plate and above and with S9: 33 µg/plate and above
TA1537: without S9: 33 µg/plate and above and with S9: 66 µg/plate and above
TA98: without S9: 33 µg/plate and above and with S9: 33 µg/plate and above
TA100: without S9: 33 µg/plate and above and with S9: 33 µg/plate and above
WP2uvrA: without S9: 100 µg/plate and above and with S9: 200 µg/plate and above
Conclusions:
Based on the results of this study it is concluded that Oleyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of Oleyl tripropylenetetramine in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

 

Oleyl tripropylenetetramine was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

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

 

Batch S001029 of Oleyl tripropylenetetramine was a white paste. The test substance was dissolved in ethanol. Table 1 gives an overview of the experiments conducted.

 

Table 1 Overview of experiments

Experiment

Tester strains

Metabolic activation

Concentration range (µg/plate)

Dose range finding

TA100 and WP2uvrA

-

5% (v/v) S9

Up to 5000 µg/plate

Up to 5000 µg/plate

Experiment 1

TA1535, TA1537, TA98 and TA100

-

5% (v/v) S9

Up to 66 µg/plate

Up to 66 µg/plate

Experiment 2

TA1535

-

10% (v/v) S9

Up to 33 µg/plate

Up to 33 µg/plate

TA1537, TA98 and TA100

-

10% (v/v) S9

Up to 66 µg/plate

Up to 66 µg/plate

WP2uvrA

-

10% (v/v) S9

Up to 200 µg/plate

Up to 200 µg/plate

-  no S9-mix added.

 

Oleyl tripropylenetetramine precipitated on the plates at the top dose of 5000 µg/plate.

Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the

bacterial background lawn, was observed in all tester strains in the absence and presence of S9-

mix.

 

Oleyl tripropylenetetramine did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Oleyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April 2016 (set up of blood cultures) and 20 June 2016 (last day of slide analysis)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 26 September 2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Substance as described in Chapter 1.1
Target gene:
Chromosome aberration
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Blood samples were obtained by venapuncture from three young healthy, non-smoking individuals (30, 36 and 33 years old) with no known recent exposures to genotoxic chemicals or radiation.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver (S9 mix); final concentration in culture medium was 4%.
Test concentrations with justification for top dose:
Preliminary tests: 5000, 2500, 1250, 625, 313, 156, 78, 39, 20 and 10 μg/ml. (solutions visually and pH checked)
First experiment: 2500, 1250, 625, 313, 156, 78, 39, 20 and 10 μg/ml - pulse, with and without metabolic activation
second experiment: 300, 250, 200, 150, 100, 75, 50, 30, 15 and 7.5 μg/ml - pulse, with and without metabolic activation
third experiment: 30, 25, 20, 15, 10, 7.5, 5.0, 3.0, 1.0, 0.5 and 0.25 μg/ml - continuous treatment and pulse, without metabolic activation.
fourth experiment: 45, 40, 35, 30, 25, 20, 15, 10 and 5.0 μg/ml - continuous treatment.
Vehicle / solvent:
DMSO was used as vehicle for stock solutions.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Vinblastine sulphate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hr, in the presence of phytoheamagglutinine (PHA-L)
- Exposure duration:
Short-term (pulse) treatment: Without and with S9-mix: 4 hr treatment, plus 20 hr recovery/harvest time
Continuous treatment: Without S9-mix: 24 hr treatment/harvest time
ARREST OF CELL DIVISION: 5 μg/mL Cytochalasine B from start of exposure
SLIDES: The cells were harvested by low speed centrifugation, briefly treated with a hypotonic solution (0.075 M potassium chloride), fixed three times with a freshly prepared mixture of methanol and acetic acid, spread on clean slides and air dried. Three slides were prepared from each selected culture
STAIN: fluorescence DNAspecific dye (acridin-orange)
NUMBER OF REPLICATIONS: duplicates
SCORIG: "blind" scoring; One slide per culture was analysed for Cytokinesis-Block Proliferation Index (CBPI) and two slides were analysed for micronucleus formation.
NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)
DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)
Evaluation criteria:
The frequencies of micronuclei in the treated cultures were compared with those of the concurrent solvent control.
The study was considered valid if the clastogenic and aneugenic positive controls gave a statistically significant increase in the number of binucleated cells containing micronuclei and if the solvent controls (DMSO) were within the historical data of the test facility.

The response was considered positive if all of the following criteria are met:
- at least one of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control.
- the increase is dose-related in at least in one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical solvent control data.

A response was considered negative if all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control.
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data.

A test substance was considered equivocal if the response was neither positive or negative even after further investigation.

Statistical methods were used as an aid in evaluating the test results. Both biological relevance and statistical analysis were considered in evaluation of the response. Biological relevance was evaluated by comparison of the test results with the test facility’s historical range of the solvent control.
Statistics:
The frequencies micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using the Chi-square test (one-sided). The results were considered statistically significant when the p-value of the Chi-square test was less than 0.05.
Key result
Species / strain:
lymphocytes:
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
Additional information on results:
PRILIMINARY TESTS
In the solubility test, it was observed that DMSO was a suitable vehicle for the test substance.
For UVCBs the maximum concentration in the final culture medium should be 5 mg/ml.
After preparation of the final concentrations (5000, 2500, 1250 and 625 μg/ml) in the culture medium without serum, a dose related turbidity and precipitation of the test substance was observed. At the highest two test substance concentrations, the culture medium appeared to be purple compared to control cultures. At the lower (78 to 313 μg/ml) concentrations no aberrant findings were observed.
In culture medium, at the highest two concentrations the pH value was somewhat increased when compared to the control cultures (pH 8.33, 7.92 and 7.25 for 5000 μg/ml, 2500 μg/ml and solvent control, respectively).
The osmolality values of the final concentrations were not determined as no increase in osmolality was expected.

CONTROLS
In the conducted experiments, the solvent controls (1% and 2% DMSO) were within the range of historical data of the test facility. Treatment with the positive control substances Cyclophosphamide, Mitomycin C and Vinblastine Sulphate resulted in statistically significant increases in the number of binucleated cells containing micronuclei, when compared to the numbers found in the concurrent solvent control cultures. This demonstrated the validity of the in vitro micronucleus test

CYTOTOXICITY
The first experiment was terminated without performing the Cytokinesis-Block Proliferation Index (CBPI) and micronuclei analysis due to the low quality of the slides.
In the pulse treatment group with S9-mix (second experiment), the test substance showed a concentration related cytotoxicity. The test substance concentrations of 300 to 75 μg/ml were severely cytotoxic to the cells, as demonstrated by the absence of cells on the slides. The test substance concentration of 50 μg/ml was strongly cytotoxic. The next lower concentrations (30, 15 and 7.5 μg/ml) showed 50%, 27% and 11% cytotoxicity, respectively. In addition, at the concentration of 30 μg/ml a low cell density on the slides was observed. The positive control substance Cyclophosphamide (20 μg/ml) showed 66% cytotoxicity.

In the pulse treatment group without S9-mix (second experiment), the test substance concentrations of 300 to 15 μg/ml were severely cytotoxic to the cells, as demonstrated by the absence of cells observed during slide preparation (visual inspection under microscope; CBPI was not determined). The cytotoxicity appeared to be more severe than was expected based on the observations of the first experiment. As a consequence, three analyzable test substance concentrations were not available and this pulse treatment group was repeated in the third experiment.
In the repeated pulse treatment group without S9-mix (third experiment), the test substance demonstrated a concentration related cytotoxicity. The test substance concentration of 30 μg/ml was severely cytotoxic to the cells, demonstrated by the absence of cells. The next lower concentrations (25, 20 and 15 μg/ml) showed 41%, 38% and 24% cytotoxicity, respectively. In addition, at the concentrations analyzed a low cell density on the slides was observed. The lowest test substance concentrations (0.5 to 10 μg/ml) did not show any cytotoxicity when compared to the concurrent control cultures. The positive control substance Mitomycine C (0.4μg/ml) showed 58% cytotoxicity.
In the continuous treatment group without S9-mix (third experiment), at the highest concentration (30 μg/ml) the test substance induced 33% cytotoxicity. In addition, at this concentration a low cell density on the slides was observed. At the lower concentrations (0.5 to 25 μg/ml) the cytotoxicity fluctuated between 9% to 24%, when compared to the concurrent solvent control. As the aimed cytotoxicity of 55±5% was not be achieved at the highest concentration tested, this treatment group was repeated with higher dose levels in a fourth experiment. The positive control substance Vinblastine sulphate (0.0125μg/ml) showed 49% cytotoxicity.
In the repeated continuous treatment group without S9-mix (fourth experiment), at the highest concentration (45 μg/ml) the test substance appeared to be severely cytotoxic to the cells. The next lower concentration (40 μg/ml) showed a cytotoxicity of 26% based on the CBPI. In addition, during the analysis, cell debris, low cell density and occasionally fragmented nuclei and affected cytoplasm was observed on the slides. These findings indicate that the actual cytotoxicity appeared to be higher than the cytotoxicity calculated based on the CBPI. At the concentration of 35 μg/ml, the cytotoxicity was 21% based on the CBPI, but the same visual observations were made as for 40 μg/ml. At the concentration of 30 μg/ml, cell density was reduced and cell debris was observed. At the lowest dose levels (10 to 25 μg/ml) cytotoxicity fluctuated between 12% and 16%, when compared to the concurrent solvent control. The positive control substance Vinblastine sulphate (0.0125μg/ml) showed 71% cytotoxicity.

The first experiment was terminated without performing the Cytokinesis-Block Proliferation Index (CBPI) and micronuclei analysis due to the low quality of the slides.
In the pulse treatment group with S9-mix (second experiment), the test substance showed a concentration related cytotoxicity. The test substance concentrations of 300 to 75 μg/ml were severely cytotoxic to the cells, as demonstrated by the absence of cells on the slides. The test substance concentration of 50 μg/ml was strongly cytotoxic. The next lower concentrations (30, 15 and 7.5 μg/ml) showed 50%, 27% and 11% cytotoxicity, respectively. In addition, at the concentration of 30 μg/ml a low cell density on the slides was observed. The positive control substance Cyclophosphamide (20 μg/ml) showed 66% cytotoxicity.

In the pulse treatment group without S9-mix (second experiment), the test substance concentrations of 300 to 15 μg/ml were severely cytotoxic to the cells, as demonstrated by the absence of cells observed during slide preparation (visual inspection under microscope; CBPI was not determined). The cytotoxicity appeared to be more severe than was expected based on the observations of the first experiment. As a consequence, three analyzable test substance concentrations were not available and this pulse treatment group was repeated in the third experiment.
In the repeated pulse treatment group without S9-mix (third experiment), the test substance demonstrated a concentration related cytotoxicity. The test substance concentration of 30 μg/ml was severely cytotoxic to the cells, demonstrated by the absence of cells. The next lower concentrations (25, 20 and 15 μg/ml) showed 41%, 38% and 24% cytotoxicity, respectively. In addition, at the concentrations analyzed a low cell density on the slides was observed. The lowest test substance concentrations (0.5 to 10 μg/ml) did not show any cytotoxicity when compared to the concurrent control cultures. The positive control substance Mitomycine C (0.4μg/ml) showed 58% cytotoxicity.
In the continuous treatment group without S9-mix (third experiment), at the highest concentration (30 μg/ml) the test substance induced 33% cytotoxicity. In addition, at this concentration a low cell density on the slides was observed. At the lower concentrations (0.5 to 25 μg/ml) the cytotoxicity fluctuated between 9% to 24%, when compared to the concurrent solvent control. As the aimed cytotoxicity of 55±5% was not be achieved at the highest concentration tested, this treatment group was repeated with higher dose levels in a fourth experiment. The positive control substance Vinblastine sulphate (0.0125μg/ml) showed 49% cytotoxicity.
In the repeated continuous treatment group without S9-mix (fourth experiment), at the highest concentration (45 μg/ml) the test substance appeared to be severely cytotoxic to the cells. The next lower concentration (40 μg/ml) showed a cytotoxicity of 26% based on the CBPI. In addition, during the analysis, cell debris, low cell density and occasionally fragmented nuclei and affected cytoplasm was observed on the slides. These findings indicate that the actual cytotoxicity appeared to be higher than the cytotoxicity calculated based on the CBPI. At the concentration of 35 μg/ml, the cytotoxicity was 21% based on the CBPI, but the same visual observations were made as for 40 μg/ml. At the concentration of 30 μg/ml, cell density was reduced and cell debris was observed. At the lowest dose levels (10 to 25 μg/ml) cytotoxicity fluctuated between 12% and 16%, when compared to the concurrent solvent control. The positive control substance Vinblastine sulphate (0.0125μg/ml) showed 71% cytotoxicity.

MICRONUCLEI INDUCTION
Since historical solvent control data for final concentrations of 1% and 2% DMSO in the continuous treatment group when co-exposed with cytochalasin B (requirement of the updated OECD test guideline 487) were limited at the test facility, the numbers of binucleated cells containing a micronucleus were not only compared with the historical data at the test facility but also with data presented in the literature (M. Fenech et al, 2003 and E. Lorge et al, 2006). The number of binucleated cells containing micronuclei were within the historical data range of the test facility and published literature
Remarks on result:
other: Both 4 h (pulse, with and without S9) treatment and 20 h (continuous) treatment were negative.

Experiment 2►Pulse treatment method with metabolic activation

Treatm / recovery time (h) Dose level (μg/ml) Cell stage analysis/500 (MO-BN-MU) BN (%) CBPI CBPI (mean) RI (%) % Cytotox. (100-RI) Selected for MN analysis (+/-) MNBN/ 1000BN MNBN/ 2000BN (%) Statistics1(p-value)
4/20 (+S9) NC 237 260 3 52.0 1.532 1.536 100 0 + 10 20
(1.00)
-
231 268 1 53.6 1.540 10
300 *    *    * - - - - - - - - -
*    *    * - - -
250 *    *    * - - - - - - - - -
*    *    * - - -
200 *    *    * - - - - - - - - -
*    *    * - - -
150 *    *    * - - - - - - - - -
*    *    * - - -
100 *    *    * - - - - - - - - -
*    *    * - - -
75 *    *    * - - - - - - - - -
*    *    * - - -
50 *    *    * - - - -
13
-
87
- - - -
464  36  0 7.2 1.077 -
30 371 129 0 25.8  1.258 1.266 50 50 + 9 21
(1.05)
n.s.
363 137 0 27.4 1.274 12
15 302 194 4 38.8  1.404 1.390 73 27 + 9 16
(0.80)
n.s.
313 186 1 37.2  1.376 7
7.5 264 234 2 46.8  1.476 1.479 89 11 + 10 17
(0.85)
n.s.
263 233 4 46.6  1.482 7
CP 20 407 93 0 18.6  1.186 1.184 34 66 + 26 54
(2.70)
<0.0001
***
409 91 0 18.2  1.182 28

Abbreviations:

Treatm: treatment time

Cytotox: cytotoxicity

MO: Mononucleated Cells

BN: Binucleated Cells

MU: Multinucleated Cells

CBPI: Cytokinesis-Block Proliferation Index

RI: Replication index

MN: Micronuclei

MNBN: Micronucleated Binucleated Cells

NC: negative control (culture medium)

- : not selected

* : No cells available on the slides

CP: Cyclophosphamide

1) Chi-square test (one-sided); *** p≤0.0001

n.s: not significant compared to the concurrent control

Experiment 3►Pulse treatment method without metabolic activation

Treatm / recovery time (h) Dose level (μg/ml) Cell stage analysis/500 (MO-BN-MU) BN (%) CBPI CBPI (mean) RI (%) % Cytotox. (100-RI) Selected for MN analysis (+/-) MNBN/ 1000BN MNBN/ 2000BN (%) Statistics1(p-value)
4/20 (-S9) NC 194 296 10 59.2  1.632 1.602 100 0 + 12 19
(0.95_
-
220 274 6 54.8  1.572 10
30 *    *    * - - - - - - - - -
*    *    * - - -
25 345 155 0 31.0 1.310 1.355 59 41 + 10 21 (1.05)  n.s.
300 200 0 40 1.400 11
20 307 192 1 38.4 1.388 1.374 62 38 - - - -
320 180 0 36 1.360 -
15 285 212 3 42.4 1.436 1.456 76 24 + 12 22 (1.10)  n.s.
265 232 3 46.4 1.476 10
10 252 240 8 48.0 1.512 1.54 90 10 - - - -
221 274 5 54.8 1.568 -
7.5 246 247 7 49.4 1.522 1.548 91 9 - - - -
220 273 7 54.6 1.574 -
5.0 212 282 6 56.4 1.588 1.558 93 7 + 10 19 (0.95)  n.s.
243 250 7 50 1.528 9
3.0 226 262 12 52.4 1.572 1.6 100 0 - - - -
195 296 9 59.2 1.628 -
1.0 204 291 5 58.2 1.602 1.605 100 0 - - - -
205 286 9 57.2 1.608 -
0.5 192 299 9 59.8 1.634 1.62 103 0 - - - -
210 277 13 55.4 1.606 -
MMC 0.4 368 131 1 26.2 1.266 1.251 42 58 + 71 136
(6.80)
<0.0001
***
382 118 0 23.6 1.236 65

The fixed cells of dose level 0.25 μg/ml were stored without slide preparation.

Abbreviations:

Treatm: treatment time

Cytotox: cytotoxicity

MO: Mononucleated Cells

BN: Binucleated Cells

MU: Multinucleated Cells

CBPI: Cytokinesis-Block Proliferation Index

RI: Replication index

MN: Micronuclei

MNBN: Micronucleated Binucleated Cells

NC: negative control (culture medium)

- : not selected

* : No cells available on the slides

MMC: Mitomycin C

1) Chi-square test (one-sided); *** p≤0.0001

n.s: not significant compared to the concurrent control

Experiment 3►Continuous treatment method without metabolic activation

Treatm / recovery time (h) Dose level (μg/ml) Cell stage analysis/500 (MO-BN-MU) BN (%) CBPI CBPI (mean) RI (%) % Cytotox. (100-RI) Selected for MN analysis (+/-) MNBN/ 1000BN MNBN/ 2000BN (%) Statistics1(p-value)
24h (-S9) NC 209 278 13  55.6 1.608 1.609 100 0 - - - -
207 281 12  56.2 1.610 -
NC* 260 226 14  45.2 1.508 1.529 87 13 - - - -
236 253 11  50.6 1.550 -
30 299 199 2  39.8 1.406 1.407 67 33 - - - -
302 192 6  38.4 1.408 -
25 299 194 7  38.8 1.416 1.46 76 24 - - - -
257 234 9  46.8 1.504 -
20 266 229 5  45.8 1.478 1.469 77 23 - - - -
279 212 9  42.4 1.460 -
15 285 210 5  42.0 1.440 1.477 78 22 - - - -
249 245 6  49.0 1.514 -
10 225 262 13  52.4 1.576 1.554 91 9 - - - -
250 234 16  46.8 1.532 -
7.5 242 246 12  49.2 1.540 1.503 83 17 - - - -
270 227 3  45.4 1.466 -
5.0 289 207 4  41.4 1.430 1.463 76 24 - - - -
259 234 7  46.8 1.496 -
3.0 261 232 7  46.4 1.492 1.516 85 15 - - - -
241 248 11  49.6 1.540 -
1.0 256 236 8  47.2 1.504 1.538 88 12 - - - -
223 268 9  53.6 1.572 -
0.5 243 247 10  49.4 1.534 1.510 84 16 - - - -
264 229 7  45.8 1.486 -
VB 0.0125 351 136 13  27.2 1.324 1.310 51 49 - - - -
357 138 5  27.6 1.296 -

The fixed cells of dose level 0.25 μg/ml were stored without slide preparation

Abbreviations:

Treatm: treatment time, Cytotox: cytotoxicity

MO: Mononucleated Cells, BN: Binucleated Cells, MU: Multinucleated Cells

CBPI: Cytokinesis-Block Proliferation Index

RI: Replication index, MN: Micronuclei, MNBN: Micronucleated Binucleated Cells

NC: negative control (2% DMSO), NC#: negative control (1% DMSO)

VB: Vinblastine sulphate,

-: Not selected / determined

1) Chi-square test (one-sided)

Experiment 4►Continuous treatment method without metabolic activation

Treatm / recovery time (h)

Dose level

(μg/ml)

Cell stage analysis/500

(MO-BN-MU)

BN

(%)

CBPI

CBPI

(mean)

RI

(%)

% Cytotox.

(100-RI)

Selected

for MN analysis (+/-)

MNBN/ 1000BN

MNBN/

2000BN (%)

Statistics1(p-value)
24h (-S9) NC 258 230 12 46.0 1.508 1.526 100 0 +  8

17 (0.85) 

-
240  248 12 49.6 1.544 9
NC* 254  232 14 46.4 1.520 1.551 105 0 +  8

17

(0.85) 

-
228 253 19 50.6 1.582 9
45 *    *    * - - - - - - - - -
*    *    * - -  
40 308  192  0 38.4 1.384 1.388 74 26 +  9

19

(0.95) 

n.s.
308  188  4 37.6 1.392 10
35 301  194  5 38.8 1.408 1.414 79 21 - - - -
294  202  4 40.4 1.420 -
30 269  227  4 45.4 1.470 1.474 90 10 - - - -
269  223  8 44.6 1.478 -
25 291  203  6 40.6 1.430 1.444 84 16 +  11

20

(1.00) 

n.s.
281  209 10 41.8 1.458 9
20 269  218 13 43.6 1.488 1.462 88 12 - - - -
291  200  9 40.0 1.436 -
15 304  191  5 38.2 1.402 1.443 84 16 - - - -
274  210 16 42.0 1.484 -
10 301  192  7 38.4 1.412 1.442 84 16 - - - -
271  222  7 44.4 1.472 -
5 248  244  8 48.8 1.520 1.543 103 0 +  9

17

(0.85) 

n.s.
227  263 10 52.6 1.566 8
VB 0.125 436  61  3 12.2 1.134 1.151 29 71 +  61

112

(5.60) 

<0.0001
***
422  72  6 14.4 1.168 51

The fixed cells of dose level 0.25 μg/ml were stored without slide preparation

Abbreviations:

Treatm: treatment time, Cytotox: cytotoxicity

MO: Mononucleated Cells, BN: Binucleated Cells, MU: Multinucleated Cells

CBPI: Cytokinesis-Block Proliferation Index

RI: Replication index, MN: Micronuclei, MNBN: Micronucleated Binucleated Cells

NC: negative control (2% DMSO), NC#: negative control (1% DMSO)

VB: Vinblastine sulphate, * No cells available on the slides

- : Not selected / determined

1) Chi-square test (one-sided); *** p≤0.0001

Conclusions:
Oley(vegetable oil) tripropylenetetramine was not clastogenic and/or aneugenic to cultured human lymphocytes.
Executive summary:

The test substance, Oleyl(vegetable oil) tripropylenetetramine was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix).

Four independent experiments were conducted for which blood was obtained from three different donors. Dimethylsufoxide (DMSO) was used as a solvent for the test substance. The final concentrations of the test substance in the cultures ranged from 0.25 to 2500 μg/ml. In the first test, the maximum feasible final concentration in the culture medium was 2500 μg/ml, based on the solubility of the test substance. Duplicate cultures were used in all experiments. Cytotoxicity was determined from the Cytokinesis-Block Proliferation Index (CBPI) and from observed concurrent measures (such as signs of apoptosis or necrosis and low cell density on the slides).

In the first test, in the presence and absence of S9-mix the treatment/recovery time was 4/20 hours (pulse treatment). In the second test, in the continuous treatment group the treatment/recovery time was 24/0 hours. Solvent control (DMSO) and positive controls were run in parallel.

 

In all four experiments, the solvent controls (1% and 2% DMSO) were within the range of the historical data of the test facility and comparable to the data presented in the literature. Treatment with the positive controls Cyclophosphamide, Mitomycine C and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the solvent control in all experiments. This demonstrates the validity of the study.

 

The first experiment was terminated without performing the Cytokinesis-Block Proliferation Index (CBPI) and micronuclei analysis due to the low quality of the slides. Hence, the incubations for the pulse treatment groups both with and without S9-mix were repeated.

In the repeated pulse treatment group with S9-mix (second experiment), the test substance showed a concentration related cytotoxicity. Test substance concentrations of 300 to 75 μg/ml appeared to be severely cytotoxic to the cells, as demonstrated by the absence of cells on the slides. The concentrations selected for analysis of micronucleus induction (30, 15 and 7.5 μg/ml) showed 50%, 27% and 11% cytotoxicity, respectively. In the pulse treatment group without S9-mix (second experiment), the cytotoxicity appeared to be more severe than it was expected based on the observations of the first experiment. As a consequence, three analyzable test substance concentrations were not available and this pulse treatment group was repeated.

In the repeated pulse treatment group without S9-mix (third experiment), the test substance showed a concentration related cytotoxicity. The test substance concentration of 30 μg/ml was severely cytotoxic to the cells, as demonstrated by the absence of cells. The concentrations selected for analysis of micronucleus induction (25, 15 and 5 μg/ml) showed 41%, 24% and 7% cytotoxicity, respectively. The concentration response relationship with respect to cytotoxicity appeared to be very steep as the concentrations were already closely spaced and the next higher concentration (30 μg/ml) was severely cytotoxic to the cells. Therefore, the concentration of 25 μg/ml with 41% cytotoxicity was considered acceptable and selected as the highest concentration for microscopic evaluation. In both the pulse treatment groups, the test substance did not show a statistically significant, dose-depended increase in the number of binucleated cells containing micronuclei at any of the concentrations analyzed when compared to the concurrent solvent cultures. In the continuous treatment group without S9-mix (third experiment), at the highest concentration (30 μg/ml) the test substance induced 30% cytotoxicity. As the aimed cytotoxicity of 55±5% was not be achieved at the highest concentration tested, this treatment group was repeated with higher dose levels in a fourth experiment.

In the repeated continuous treatment group without S9-mix (fourth experiment), at the highest concentration (45 μg/ml) the test substance appeared to be severely cytotoxic to the cells. The next lower concentration (40 μg/ml) showed a cytotoxicity of 26% based on the CBPI. The concentrations selected for analysis of micronucleus induction (40, 25 and 5 μg/ml) showed a cytotoxicity of 26%, 16% and 0%, respectively. Although the cytotoxicity of the highest concentration selected was lower than the aimed cytotoxicity level of 55±5% as stated in the study plan, the presence of cell debris, low cell density and occasionally fragmented nuclei and affected cytoplasm was observed on the slides during the microscopic analysis. These observations indicate that the actual cytotoxicity appeared to be higher than the cytotoxicity calculated based on the CBPI. In addition, the dose response relationship with respect to cytotoxicity appeared to be very steep, as a concentration of 45 μg/ml was severely cytotoxic to the cells. Therefore, the concentration of 40 μg/ml with 26% cytotoxicity was considered acceptable and selected as the highest concentration for microscopic evaluation. In this treatment group, the test substance did not show a statistically significant, dose-depended increase in the number of binucleated cells containing micronuclei at any of the concentrations analyzed when compared to the concurrent solvent cultures.

 

From the results obtained in the in vitro micronucleus test it is concluded that, under the conditions used in this study, the test substance Oleyl(vegetable oil) tripropylenetetramine was not clastogenic and/or aneugenic to cultured human lymphocytes.

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:
24-jun-2009 to 17-aug-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Cross-reading from this substance is acceptable on the basis of identical alkyl-tripropylenetetramine structure, resulting to the same functional groups with similar properties leading to common biological activity, and common metabolic degradation. Oleyl tripropylene tetramine is a linear tetramine and can be compared to the already registered Tallow tripropylene tetramine, where the average chain length for Oleyl is marginally higher, as well as the level of unsaturation. (See table below).
As both a lower chain length and a higher unsaturation, are generally linked to an increased toxicity compared to longer chain length and saturation. All in all, in is not expected that both substances will differ much from each other.
Further information on the applicability of the read-across can be obtained from the document "Category polyamines - 20170518.pdf" added to IUCLID Ch. 13.

Common chemical name: Oleyl tripropylene tetramine
- CAS: 67228-83-5
- Name (EC): Oleyl(vegetable oil) tripropylene tetramine
Common chemical name: Tallow tripropylene tetramine
- CAS: 1219458-11-3
- Name (EC): Tetraamine C16-18, C18-unsaturated

Composition: Tallow Oleyl
tetramine % 35-65 42-47
triamine % 15-37 30-37
diamine % 8-12 < 12
primary amine % 2-6 < 4
higher amines % < 10 < 10
Iodine g/100g 25-47 35-55

chain length distribution in representative products:
Tallow Oleyl
C18:1 40 41
C18 26 51
C16 34 8
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.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: 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)
Deviations:
no
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):
- 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 (Sigma) and 2 mM L-glutamin (Invitrogen Corporation) 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: 0.3, 1, 3, 10, 33, 100 and 333 µg/ml
Without S9-mix, 24 hours treatment: 0.1, 0.3, 1, 3, 10, 33 and 100 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.25, 0.5, 1, 1.5, 2, 2.5, 3 and 3.5 μg/ml
With S9-mix, 3 hours treatment: 1, 10, 14, 16, 18, 20, 22 and 24 μg/ml
Experiment 2
Without S9-mix, 24 hours treatment: 0.4, 0.8, 1.2, 1.6, 2, 2.4, 2.8 and 3.2 µg/ml
With S9-mix, 3 hours treatment: 10, 15, 20, 25, 30, 32.5, 35 and 37.5 μg/ml

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Stability in ethanol is known and ethanol is 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 105 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 (ref. 12).

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.
Key result
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 100 µg/ml and above at the start of the treatment.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 µg/ml in the absence of S9, 3 hours treatment; at dose levels of 33 µg/ml in the presence of S9, 3 hours treatment; at dose levels of 10 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 79 and 83% 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 85 and 73% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment , respectively.
Conclusions:
It is concluded that Tallow tripropylenetetramine is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

Tallow tripropylenetetramine 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.

 

Batch S001255 of Tallow tripropylenetetramine was a white paste. The test substance was dissolved in ethanol.

 

In the first experiment, Tallow tripropylenetetramine was tested up to concentrations of 3.5 and 24 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Tallow tripropylenetetramine was tested up to cytotoxic levels of 79 and 85% in the absence and presence of S9-mix, respectively.

In the second experiment, Tallow tripropylenetetramine was tested up to concentrations of 3.2 and 37.5 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. Tallow tripropylenetetramine was tested up to cytotoxic levels of 83% in the absence of S9-mix and up to 73% 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

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, Tallow tripropylenetetramine 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, Tallow tripropylenetetramine 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 Tallow tripropylenetetramine 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

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genotoxicity by alkyl-tripropylenetetramines is not expected. In physiological circumstances, these polyamines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil.

 

Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

Oleyl tripropylenetetramine was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of Phenobarbital and ß-naphthoflavone). 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 Oleyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Oley tripropylenetetramine was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix). The study was performed under GLP and according to the most recent OECD and EU guidelines. From the results obtained in the in vitro micronucleus test it is concluded that, under the conditions used in this study, the test substance Oleyl tripropylenetetramine was not clastogenic and/or aneugenic to cultured human lymphocytes.

 

Finally, in vitro mammalian mutagenicity was evaluated with the similar substance Tallow (C16-18, C18-unsat.) tripropylenetetramine. Tallow tripropylenetetramine 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,Tallow tripropylenetetramine 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, Tallow tripropylenetetraamine is not mutagenic in the TK mutation test.

 

Additionally, polyamines do not react with DNA or react to protein (indicated by OECD Toolbox profiling). See also the document "Category polyamines - 20170518.pdf" added to IUCLID Ch. 13.

Justification for classification or non-classification

Oleyl tripropylenetetramine is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay, is not clastogenic in human lymphocytes, and by means of cross-reading to Tallow (C16-18, C18 unsat.) tripropylenetetramine, also not mutagenic in the TK mutation test with L5178Y mouse lymphoma cells.

Furthermore, polyamines do not react with DNA or react to protein.

 

As the substance shows no evidence for genotoxicity, no classification for genotoxic effects is required.