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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a GLP compliant study is available on the basis of testing withOleyl dipropylene triamineitself or from cross-reading from Tallow dipropylenetriamine.Lack of genotoxic properties of alkyl-dipropylenetriamines was further confirmed inin vitrostudies on Coco dipropylene triamine. Mode of action analysis further shows that genotoxic properties are not likely.
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
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 S9-mix: 0.3, 1, 3, 10, 20 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
Experiment 2:
TA1535, TA1537, TA98 and TA100
(Without S9): 0.3, 1, 3, 10, 20 and 33 µg/plate
(With S9): 0.3, 1, 3, 10, 33 and 66 µg/plate
WP2uvrA:
(Without and with S9): 1, 3, 10, 33, 66 and 100 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 tester 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
- Method: 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 test substance
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).
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitate 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 at 100 µg/plate in the absence and 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: 20 µg/plate and above and with S9: 33 µg/plate and above
TA1537: without S9: 20 µg/plate and above and with S9: 10 µg/plate and above
TA98: without S9: 20 µg/plate and above and with S9: 33 µg/plate and above
TA100: without S9: 20 µg/plate and above and with S9: 33 µg/plate and above
WP2uvrA: without S9: 66 µg/plate and above and with S9: 100 µg/plate and above

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

Oleyl dipropylenetriamine 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.

Oleyl dipropylenetriamine was an off white turbid liquid. The test substance was dissolved in ethanol.

 

Experiment

Tester strains

Metabolic activation

Concentration range

Dose range finding

TA100 and WP2uvrA

-

5% (v/v) S9

Up to 5000μg/plate

Up to 5000μg/plate

Experiment 1

TA1535, TA1537, TA98 and TA100

-

5% (v/v) S9

Up to 33μg/plate

Up to 66μg/plate

Experiment 2

TA1535, TA1537, TA98 and TA100

-

10% (v/v) S9

Up to 33μg/plate

Up to 66μg/plate

WP2uvrA

-

10% (v/v) S9

Up to 100μg/plate

Up to 100μg/plate

- no S9-mix added.

Oleyl dipropylenetriamine 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 dipropylenetriamine 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 dipropylenetriamine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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:
24-jun-2009 to 12-sep-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
(See also Category polyamines in support of Oleyl dipropylenetriamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Tallow (C16-18, C18-unsaturated) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical 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 by the 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 metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity.
The ratio between the C16, C18 and C18:1 (C18-unsaturated) alkyl chains in the Oleyl and Tallow dipropylene triamine products show an overlap in alkyl chain of about 50%:
- Oleyl: C18:1 = 85.5%; C18 = 12.4 %; C16: 7,1%
- Tallow: C18:1 = 26.5%; C18 = 38.3 %; C16: 35.2%
The higher level of unsaturation in Oleyl based products has never shown to have an important effect. This is in agreement with the expectation that these structures do not undergo an important level of metabolism, and if metabolism occurs on the limited absorbed material, the resulting alkyl chains will fit in the physiological pool of these natural alkyl chains. Further, the relatively somewhat sorter chain lengths of C16 would make it slightly more bioavailable, in agreement to the notion that shorter alkyl chains represent a more conservative evaluation.
Consequently, the data on Tallow (C16-18, C18-unsaturated-alkyl) dipropylene triamine can be used for the read-across to Oleyl dipropylene triamine.
Reason / purpose:
read-across source
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 and with S9-mix, 3hr exposure; 24 hr fixation: 5, 10, 20, 30 and 40 µg/ml
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33, 100 and 333 µg/ml
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 14, 16 and 18 µg/ml
With S9-mix, 3 h exposure, 24 h fixation time: 10, 20 and 26 µg/ml
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 1, 10 and 15 µg/ml
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 3 and 10 µg/ml
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 22 and 26 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
Untreated negative 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

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 20 µg/ml and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 3 µg/ml and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 10 µg/ml and above in the absence of S9 for the continuous treatment of 48 hr

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.
Conclusions:
Tallow dipropylenetriamine 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.
Executive summary:

Tallow dipropylenetriamine 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 performedunder GLP and according to the most recent OECD and EU guidelines.

 

Tallow dipropylenetriamine was a white paste. Tallow dipropylenetriamine was dissolved in ethanol.

 

In the first cytogenetic assay, Tallow dipropylenetriamine was tested up to 18 and 26 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, Tallow dipropylenetriamine was tested up to 15 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 10 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 Tallow dipropylenetriamine was tested up to 26 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.

Tallow dipropylenetriamine 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 Tallow dipropylenetriamine 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. In the second cytogenetic assay the increase was dose dependent both in the absence and presence of S9-mix. This may indicate that Tallow dipropylenetriamine has the potential to inhibit mitotic processes.

No effects of Tallow dipropylenetriamine on the number of cells with endoreduplicated chromosomes was observed both in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that Tallow dipropylenetriamine is not clastogenic in human lymphocytes under the experimental conditions described in this report. Tallow dipropylenetriamine may have the potential to inhibit 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
01-jul-2009 to 11-aug-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
(See also Category polyamines in support of Oleyl dipropylenetriamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Tallow (C16-18, C18-unsaturated) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical 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 by the 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 metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity.
The ratio between the C16, C18 and C18:1 (C18-unsaturated) alkyl chains in the Oleyl and Tallow dipropylene triamine products show an overlap in alkyl chain of about 50%:
- Oleyl: C18:1 = 85.5%; C18 = 12.4 %; C16: 7,1%
- Tallow: C18:1 = 26.5%; C18 = 38.3 %; C16: 35.2%
The higher level of unsaturation in Oleyl based products has never shown to have an important effect. This is in agreement with the expectation that these structures do not undergo an important level of metabolism, and if metabolism occurs on the limited absorbed material, the resulting alkyl chains will fit in the physiological pool of these natural alkyl chains. Further, the relatively somewhat sorter chain lengths of C16 would make it slightly more bioavailable, in agreement to the notion that shorter alkyl chains represent a more conservative evaluation.
Consequently, the data on Tallow (C16-18, C18-unsaturated-alkyl) dipropylene triamine can be used for the read-across to Oleyl dipropylene triamine.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: 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. 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):
- 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: 0.3, 1, 3, 10, 33 and 100 µg/ml
Without S9-mix, 24 hours treatment: 0.08, 0.24, 0.8, 2.4, 8 and 26 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 1.5, 2 and 2.5 µg/ml
With S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 16, 18 and 20 µg/ml
Experiment 2
Without S9-mix, 24 hours treatment: 0.08, 0.24, 0.5, 0.8, 1, 1.3, 1.6 and 2 µg/ml
With S9-mix, 3 hours treatment: 1, 3, 6, 10, 15, 18, 20, 22, 26, 28 and 30 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound is stable in ethanol and this solvent 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
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: : 7.5 µg/plate
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.

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


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 2.4 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 80 and 89% 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 76% 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: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: L5178Y/TK+/--3.7.2C
Conclusions:
Interpretation of results (migrated information):
negative

Tallow dipropylenetriamine is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

Tallow dipropylenetriamine 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 S001096 of Tallow dipropylenetriamine was a white paste. The test substance was dissolved in ethanol.

In the first experiment, Tallow dipropylenetriamine was tested up to concentrations of 2.5 and 20 μg/ml in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. Tallow dipropylenetriamine was tested up to cytotoxic levels of 80 and 82% in the absence and presence of S9-mix, respectively.

In the second experiment, Tallow dipropylenetriamine was tested up to concentrations of 2 and 30 μg/ml in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Tallow dipropylenetriamine was tested up to cytotoxic levels of 89% in the absence of S9-mix and 76% 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 dipropylenetriamine did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Tallow dipropylenetriamine did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

In conclusion, Tallow dipropylenetriamine is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
15-jun-2009 to 12-aug-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
(See also Category polyamines in support of Oleyl dipropylenetriamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Coco (C12-18) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical 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 by the 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 metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity. In series of substances that are chemically identical but differ in length of alkyl chains, those that have shorter chain length are likely to be more bioavailable compared to those with longer chain lengths. Therefore, results from the shortest chain length, in this case Coco (C12-C18 alkyl) dipropylene triamine, can be considered a worst-case approach for the longer chain lengths, specifically Oleyl (C18, unsaturated) dipropylene triamine.
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:
Withoutand with S9-mix, 3hr exposure; 24 hr fixation: 10, 33 and 100 µg/ml
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33, 100 and 333 µg/ml
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 5, 10 and 15 µg/ml
With S9-mix, 3 h exposure, 24 h fixation time: 5, 15 and 25 µg/ml
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation:: 3, 7 and 15 µg/ml
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 3 and 7 µg/ml
With S9-mix, 3 hr exposure; 48 hr fixation: 20, 25 and 30 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 controls:
yes
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: At concentrations of 100 µg/ml and higher

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 33 µg/ml and above in the absence and presence of S9-mix, 3 hr treatment/24 hr fixation and at 10 µg/ml for the continuous treament of 24 and 48 hr in the absence of S9-mix .

- 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.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that Coco dipropylene triamine is not clastogenic in human lymphocytes under the experimental conditions described in this report. Coco dipropylene triamine may have the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
Executive summary:

Evaluation of the ability of Coco dipropylene triamine to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment).

This report describes the effect of Coco dipropylene triamine 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). The possible clastogenicity of Coco dipropylene triamine was tested in two independent experiments.

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

 

Batch S001254 of Coco dipropylene triamine was a yellow clear to turbid liquid with flakes. Coco dipropylene triamine was dissolved in ethanol.

 

In the first cytogenetic assay, Coco dipropylene triamine was tested up to 15 and 25 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, Coco dipropylene triamine was tested up to 15 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 7 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 Coco dipropylene triamine was tested up to 30 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.

 

Coco dipropylene triamine 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 Coco dipropylene triamine increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the presence of S9-mix in the second cytogenetic assay. This may indicate that Coco dipropylene triamine has the potential to inhibit mitotic processes.

 

No effects of Coco dipropylene triamine on the number of cells with endoreduplicated chromosomes was observed both in the absence and presence of S9-mix, in either of the two independently repeated experiments.

 

Finally, it is concluded that this test is valid and that Coco dipropylene triamine is not clastogenic in human lymphocytes under the experimental conditions described in this report. Coco dipropylene triamine may have the potential to inhibit 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:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
17-jun-2009 to 03-aug-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
(See also Category polyamines in support of Oleyl dipropylenetriamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Coco (C12-18) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical 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 by the 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 metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity. In series of substances that are chemically identical but differ in length of alkyl chains, those that have shorter chain length are likely to be more bioavailable compared to those with longer chain lengths. Therefore, results from the shortest chain length, in this case Coco (C12-C18 alkyl) dipropylene triamine, can be considered a worst-case approach for the longer chain lengths, specifically Oleyl (C18, unsaturated) dipropylene triamine.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
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):
- 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: 3, 10, 33, 100 and 333 µg/ml
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml and 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.003, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 2 µg/ml
With S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 12 µg/ml
Experiment 2
Without S9-mix, 24 hours treatment: 0.008, 0.024, 0.08, 0.2, 0.4, 0.08, 1.12 and 1.44 µg/ml
With S9-mix, 3 hours treatment: 0.01, 0.1, 1, 10, 12, 14, 16 and 17 μg/ml exposure medium

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:
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
.

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.
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.


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
- Other confounding effects:


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 1 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.

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

It is concluded that Coco dipropylene triamine is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

Evaluation of the mutagenic activity of Coco dipropylene triamine in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

This report describes the effects of Coco dipropylene triamine on the 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 procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch S001254 of Coco dipropylene triamine was a yellow clear to turbid liquid with flakes. The test substance was dissolved in ethanol.

 

In the first experiment, Coco dipropylene triamine was tested up to concentrations of 2 and 12 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Coco dipropylene triamine was tested up to cytotoxic levels of 78 and 69% in the absence and presence of S9-mix, respectively.

In the second experiment, Coco dipropylene triamine was tested up to concentrations of 1.44 and 17 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix. Coco dipropylene triamine was tested up to cytotoxic levels of 83 and 66% in the absence and presence of S9-mix, respectively.

 

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.

Mutation frequencies in cultures treated with positive control chemicals were increased by 14- and 17-fold for MMS in the absence of S9-mix, and by 11- and 8.9-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, Coco dipropylene triamine 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, Coco dipropylene triamine 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 Coco dipropylene triamine 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 is not expected. In physiological circumstances, the polyamines have a cationic surfactant structure (nitrogens are fully protonated)which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar hydrophobic tails are pushed out of solution and 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. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and to move to the nucleus to interact with DNA.

Additional information

Oleyl dipropylene triamine was not mutagenic in a bacterial mutagenicity study (Ames test). Comparable alkyl dipropylene triamine structures, also covering the same alkyl chain distribution, have shown that these substances do not induced chromosomal aberrations in human lymphocytes, and are not mutagenic in mammalian mutagenicity studies in mouse lymphoma cells. All studies were performed under GLP according to current guidelines.

 

The following studies on alkyl dipropylene triamine are available,

Alkyl chain

Study

Endpoint

Coco

1219458-12-4, NOTOX, 491200, 2009

OECD 471 Bacterial mutagenicity (Ames) (not in dossier)

Coco

1219458-12-4, NOTOX, 491233, 2009

OECD 473 Chromosomal aberration in HL

Coco

1219458-12-4, NOTOX, 491231, 2009

OECD 476 MLA (TK-locus in L5178Y cells)

Tallow

1219458-14-6, NOTOX, 491200, 2009

OECD 471 Bacterial mutagenicity (Ames) (not in dossier)

Tallow

1219458-14-6, NOTOX, 491205, 2009

OECD 473 Chromosomal aberration in HL

Tallow

1219458-14-6, NOTOX, 491203, 2009

OECD 476 MLA (TK-locus in L5178Y cells)

Oleyl

28872-01-7, NOTOX, 491213, 2009

OECD 471 Bacterial mutagenicity (Ames)

Coco = C12-18

Tallow = C16-18, C18-unsat.

Oleyl = C18 unsaturated

 

Oleyl dipropylene triamine 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 dipropylene triamine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Tallow dipropylenetriamine 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.

Tallow dipropylenetriamine 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 Tallow dipropylenetriamine 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. In the second cytogenetic assay the increase was dose dependent both in the absence and presence of S9-mix. Polyploidy alone does not indicate aneugenic potential and can simply indicate cell cycle perturbation; it is also commonly associated with increasing cytotoxicity.

There were no effects of Tallow dipropylenetriamine on the number of cells with endoreduplicated chromosomes both in the absence and presence of S9-mix, in either of the two independently repeated experiments. Therefore, it is concluded that Tallow dipropylenetriamine is not clastogenic in human lymphocytes.

 

Tallow dipropylenetriamine 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 dipropylenetriamine 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 dipropylenetriamine is not mutagenic in the TK mutation test.

 

Cocdipropylenetriamine 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.

Cocodipropylenetriamine 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 Coco dipropylene triamine increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the presence of S9-mix in the second cytogenetic assay. Polyploidy alone does not indicate aneugenic potential and can simply indicate cell cycle perturbation; it is also commonly associated with increasing cytotoxicity.

There were no effects of Cocodipropylenetriamine on the number of cells with endoreduplicated chromosomes both in the absence and presence of S9-mix, in either of the two independently repeated experiments. Therefore, it is concluded that Cocodipropylenetriamine is not clastogenic in human lymphocytes.

 

Cocodipropylenetriamine 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, Cocodipropylenetriamine 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, Cocodipropylenetriamine is not mutagenic in the TK mutation test.

 

Also studies on other polyamines indicate no concerns for genotoxicity. Furthermore, polyamines do not react with DNA or react to protein. Mode of action analysis further shows that these polyamines will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and to move to the nucleus to interact with DNA.

Justification for classification or non-classification

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a GLP compliant study is available on the basis of testing withOleyl dipropylene triamineitself or from cross-reading from Tallow dipropylenetriamine.Lack of genotoxic properties of alkyl-dipropylenetriamines was further confirmed inin vitrostudies on Coco dipropylene triamine. Mode of action analysis further shows that genotoxic properties are not likely.As there is sufficient evidence for lack genotoxicity of Oley dipropylene triamine, no classification for genotoxic effects is required.