Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was not mutagenic in a bacterial mutagenicity study (Ames test), not clastogenic and/or aneugenic to cultured human lymphocytes, and 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:
22-Jun-2015 to 16-Jul-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
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
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 Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 275, 492, 878, 1568, 2800 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
other: 2-nitrofluorene 10 µg/plate in DMSO for TA98
Remarks:
without S9
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: ICR-191 25 µg/plate in DMSO for TA1537
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
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 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

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 EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 and TA98 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:
a) A two-fold (TA100, WP2uvrA) or more or a three-fold (TA1535, TA1537, TA98) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
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.
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
Untreated negative controls validity:
not examined
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:Precipitation was observed at dose levels of 1568 µg/plate and upwards

RANGE-FINDING/SCREENING STUDIES:
dose range finding:
In tester strain TA100, toxicity was observed at dose level of 500 μg/plate in the absence of S9-mix. In tester strain WP2uvrA, no toxicity was
observed up to and including the top dose of 5000 µg/plate.

main 1:
No toxicity was observed up to and including the dose level of 1600 μg/plate. The test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, therefore the number of revertants of this dose level could not be determined.
main 2:
The bacterial background lawn was not reduced at any of the concentrations tested. A decrease in the number of revertants below the historical control data range was only observed in tester strain TA1535 in the absence of S9-mix at the test substance concentration of 2800 µg/plate.
No biologically relevant decrease in the number of revertants was observed in the other tester strains up to and including the dose level of 2800 μg/plate. The test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, therefore the number of revertants of this dose level could not be determined.

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, except for the response of the positive control for TA100 in the second experiment in the presence of S9-mix. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It is concluded that di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Di-C16-C18 (evennumbered) alkyl 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 5% resp. 10% S9-mix (rat liver S9-mix induced by Aroclor 1254). The study followed the most recent OECD and EU protocols and was performed under GLP.

The test substance was dissolved in ethanol. The test substance precipitated on the plates at dose levels of 1568 μg/plate and upwards. The substance was tested up to 5000 µg/plate in all strains. The bacterial background lawn was not reduced at any of the concentrations tested. Only in TA1535 cytotoxicity evidence from decrease in the number of revertants at 2800 µg/plate in absence of S9-mix. Heavy precipitation at 5000 µg/plate prohibited determination of the number of revertant colonies at this dose level.

Acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

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 di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-Jun-2015 to 23-Oct-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood
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/first cytogeneticc test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 5.4, 17 and 52 µg/mL (first cytogenetic test)
Without S9-mix, 24 exposure; 24 hr fixation: 1.7, 5.4, 17, 52 and 164 µg /mL (dose range finding)
Additional cytogenetic test (1A):
Without and with S9-mix, 3hr exposure; 27 hr fixation: 10, 20, 30, 40 and 52 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 5.4, 17 and 52 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period
Positive control substance:
other: colchicine: 0.1 µg/mL
Remarks:
without S9
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

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:
A test substance is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a)At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b)Any of the results are outside the 95% control limits of the historical control data range.

A test substance is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a)None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided,
p < 0.05) increase compared with the concurrent negative control.
b)All results are inside the 95% control limits of the negative historical control data range.
Statistics:
In case the Chi-square test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no 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 was observed at the top dose of 52 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose
COMPARISON WITH HISTORICAL CONTROL DATA:
a)The concurrent negative control data are within the 95% control limits of the distribution of the historical negative control database.
b)The concurrent positive controls induced responses that are compatible with those generated in the historical positive control database.



Table 1   Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with di-C16-C18 (evennumbered) alkyl tripropylenetetramine in the first cytogenetic assay

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

 

 

1000

1000

2000

1000

1000

2000

 

 

A

B

A+B

A

B

A+B

0

0

2

2

4

10

2

12

5.4

8

5

0

5

5

4

9

17

0

5

1

6

5

5

10

 523)

13

1

3

4

12

12

 24*

0.25 MMC-C

32

2

2

4

36

43

79***

0.1 Colch

95

55

50

105***

 282)

 82)

36***

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

 

 

1000

1000

2000

1000

1000

2000

 

 

A

B

A+B

A

B

A+B

0

0

1

1

2

6

10

16

5.4

7

2

0

2

5

8

13

17

8

1

2

3

3

7

10

 523)

9

6

1

 7*

11

10

21

15 CP

59

2

0

2

23

8

31

17.5 CP

60

3

0

3

20

18

 38**

*)    Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)   1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2)   139 and 54 binucleated cells repectively were scored for the presence of micronuclei.

3)   The test substance precipitated in the culture medium.

 

Table 1A   Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with di-C16-C18 (evennumbered) alkyl tripropylenetetramine in cytogenetic assay 1A (additional experiment)

 

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

 

 

1000

1000

2000

1000

1000

2000

 

 

A

B

A+B

A

B

A+B

0

0

2

1

3

3

5

8

10

3

3

2

5

1

3

4

 403)

1

2

0

2

6

7

13

 523)

-6

0

2

2

7

6

13

0.25 MMC-C

32

0

3

3

34

30

64***

0.1 Colch

94

29

36

65***

 52)

 12)

6

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

 

 

1000

1000

2000

1000

1000

2000

 

 

A

B

A+B

A

B

A+B

0

0

1

1

2

6

5

11

10

-6

1

3

4

10

10

20

 403)

-3

2

3

5

11

10

21*

 523)

-2

0

3

3

12

11

23*

15 CP

60

2

0

2

17

20

37***

*)    Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)   1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2)   165 and 163 binucleated cells repectively were scored for the presence of micronuclei.

3)   The test substance precipitated in the culture medium.

 

Table 2   Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with di-C16-C18 (evennumbered) alkyl tripropylenetetramine in the second cytogenetic assay

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

 

 

1000

1000

2000

1000

1000

2000

 

 

A

B

A+B

A

B

A+B

0

0

3

1

4

2

2

4

5.4

3

2

1

3

0

1

1

17

2

0

0

0

0

1

1

 523)

-1

2

1

3

0

4

4

0.15 MMC-C

43

0

1

1

22

17

39***

0.05 Colch

96

23

19

42***

 42)

22)

 6

*)    Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)   1000-1026 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2)   603 and 209 binucleates cells respectively were scored for the presence of micronuclei.

3)   The test substance precipitated in the culture medium.

An attached graph also 95% limits of the historical control data range (upper limit 10.53) is indicated.

Conclusions:
Considering the only marginal increase observed in only one of the duplicate tests at levels showing visual precipitation, the applicant evaluates the outcome as negative.
Executive summary:

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was tested in two independent experiments for possible clastogenicity and aneugenicity in anin vitromicronucleus assay in cultured peripheral human lymphocytes (OECD 487) in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The study followed the most recent OECD and EU protocols and was performed under GLP.

 

The test substance was dissolved in ethanol.

Positive control chemicals, mitomycin C, colchicine and cyclophosphamide induced appropriate responses indicating that the assayed worked properly.

Testing was done up to 52 µg/ml, based upon occurrence of precipitation. At 17 µg/ml no precipitation was seen in the 3 hr exposure experiments, but at all dose levels from 20 µg/ml precipitation was observed (“which does not interfere with the scoring”). No (considerable) cytotoxicity was observed at any evaluated dose levels.

Increased micronuclei levels were seen at:

- 3hr:

Without S9: exp.1. increase in binucleated cells above hist. control at 52 µg/ml; increase is significant in Cochran Armitage trend test; exp. 2: negative.

With S9: exp.1: a similar increase was seen as without S9 but was not significant; However, in exp.2: a positive, above hist.control, increase was observed at 40 and 52 µg/ml.

A positive increase in mononucleated cells only occurred at highest dose level in one of the duplicates of exp. 1

- 24hr:

No increases.

Conclusion: Although statistically significant increases were observed in the first cytogenetic assay and in the additional experiment and the number of mono- and binucleated cells with micronuclei was above the 95% control limits of the distribution of the historical negative control data, none of the increases observed in the first cytogenetic assay could be confirmed in the additional experiment. Visa versa, the increases observed in the additional experiment were not observed in the first cytogenetic assay. The report therefore concluded that the results of this study are equivocal.

Since each increase in the number of mono- and binucleated cells with micronuclei was only observed once, the biological relevance of these increases are doubtful and the results are considered to be questionable under the experimental conditions described in the report.

 

The attached graph shows all result (per 2000 cells), to which a the border of the significance level (P < 0.05) is added, as well as concentrations where precipitation was observed are marked:

As shown in the graph, the increase seems overall to show a dose response relation, both with and without S9. However, levels of increased number of cells with micronuclei above lowest significant level, was only seen at the tested concentrations that also resulted to precipitation. There is no difference between with or without S9 (in exp.1); The slight increase seen in the 3 hour experiments was not seen in the 24 hr experiment, but there is only one result available in the relevant concentration range showing precipitation.

 

With the knowledge that the water solubility of this substance is extremely low (< 30 µg/L; Estimations at 25°C from WSKOW v1.42 = 5.777e-012 mg/L; WATERNT  v1.01 = 6.9329e-007 mg/L) and visible precipitations from 20 µg/ml, do lead to the concern that the observed increase in micronuclei is not physiologically relevant, and possible reflect an interaction of precipitation in the evaluation.

To that should be considered that OECD 478 states “Even if cytotoxicity occurs above the lowest insoluble concentration, it is advisable to test at only one concentration inducing turbidity or with visible precipitate because artifactual effects may result from the precipitate.

Considering the presence of visual precipitation and the only marginal increase observed in only one of the duplicate tests, the applicant evaluates the outcome as negative rather than equivocal.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-Mar-2016 to 19-Apr-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
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 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: 0.52, 1.7, 5.4, 17 and 52 µg/mL
Without S9-mix, 24 hours treatment: 0.52, 1.7, 5.4, 17 and 52 µg/ml

Experiment 1
With and Without S9-mix, 3 hours treatment: 0.054, 0.17, 0.52, 1.7, 5.4, 17, 30 and 52 µg/mL
Experiment 2:
Without S9-mix, 24 hours treatment: 0.018, 0.054, 0.17, 0.52, 1.7, 5.4 and 17 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol,
- Justification for choice of solvent/vehicle:

Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cyclophosphamide 10 µg/mL
Remarks:
with S9
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: Duplicate cultures
- Treatment groups and positive control: Single cultures

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

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. 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.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP 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.

Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
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 52 µg/mL (3-hour treatment) and 17 µg/mL (24-hour treatment)

RANGE-FINDING/SCREENING STUDIES:
3-hour treatment:
In the absence of S9-mix, the relative suspension growth was 17% at the test item concentration of 52 μg/ml compared to the relative suspension growth of
the solvent control. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test item concentrations of 52 μg/ml compared to the solvent control.
24-hour treatment:
The relative suspension growth was 54% at the test item concentration of 17 μg/ml compared to the relative suspension growth of the solvent control and no cell survival was observed at the test item concentration of 52 μg/ml.


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:
First experiment:
In the absence of S9-mix, the relative total growth of the highest test item concentration was 20% compared to the total growth of the solvent controls.
In the presence of S9-mix, no severe toxicity was observed up to and including the highest tested dose level.
Second experiment:
No severe toxicity was observed up to and including the highest tested dose level.

Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
The mouse lymphoma assay was conducted according to OECD 490 guideline and GLP principles.
In conclusion, di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix,di-C16-C18 (evennumbered) alkyl tripropylenetetraminedid not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.

 

In the presence of S9-mix,di-C16-C18 (evennumbered) alkyl tripropylenetetraminedid not induce a significant increase in the mutation frequency.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was not clastogenic or aneugenic in the in vivo bone marrow micronucleus test in male and female mice.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-Mar-2016 to 17-Jun-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
-Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
-Substance type: Viscous liquid
-Physical state: liquid
-Purity: Purity/Composition 100% (UVCB substance)
-Batch/Lot number: B1; batch inspection lot no.: 890000394200
-Expiration date of the lot/batch: 22 October 2017
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: males 34.1 ± 1.8 g and females 26.9 ± 1.3 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 22.2°C
- Humidity (%): 37 - 102%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The test item was stable in corn oil for at least 5 hours and suspension could be obtained. Corn oil has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 32, 62.5, 125 and 250 mg/ml

Duration of treatment / exposure:
Treatment:
Two treatments were performed, administered at a 24-hour interval.
Frequency of treatment:
Twice
Remarks:
Doses / Concentrations:
males 62.5, 125 and 250 mg/kg bw
females 32, 62.5 and 125 mg/kg bw

Basis:
analytical conc.
No. of animals per sex per dose:
At least five animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: ip
- Doses / concentrations: 40 mg/kg body weight
Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and cover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
A test item is considered positive in the micronucleus test if:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Statistics:
ToxRat Professional v 3.2.1 was used for statistical analysis of the data.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: not clastogenic or aneugenic
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: not clastogenic or aneugenic
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
One male and one female were dosed via intraperitoneal injection with 2000 mg/kg body weight. The animals died within 4 hours after the first dosing. One male and one female dosed with 1000 and 500 mg/kg body weight and one female dosed with 250 mg/kg body weight died within 21 hours after the first dosing. Subsequently, three males and three females were dosed with 250 and 125 mg/kg body weight, respectively. The male animals showed the following toxic signs after dosing: ataxia, lethargy, rough coat, closed eyes and a hunched posture. The female animals showed the following toxic signs after dosing: lethargy, closed eyes and a hunched posture.

RESULTS OF DEFINITIVE STUDY

- Clinical signs of toxicity in test animals:
Male animals treated with 250 mg/kg body weight showed the following toxic signs after dosing: lethargy, ataxia, rough coat, hunched posture and eyes closed. Male and female animals treated with 125 mg/kg body weight showed the following toxic signs after dosing: lethargy, rough coat, hunched posture and eyes closed. Male animals treated with 62.5 mg/kg body weight showed the following toxic signs after dosing: lethargy, hunched posture and rough coat. Female animals dosed with 62.5 mg/kg body weight had hunched posture and rough coat.

No treatment related clinical signs or mortality were noted in any female animal treated with 32 mg/kg body weight. In addition, no treatment related clinical signs or mortality were noted in the control animals receiving vehicle or cyclophosphamide.

- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item.
- Ratio of PCE/NCE (for Micronucleus assay):
The groups treated with test item concentrations of 62.5 and 32 mg/kg body weight (females) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicates a lack of toxic effects of this test item on the erythropoiesis. The groups treated with 250, 125 and 62.5 mg/kg body weight (males) and 125 mg/kg body weight (females) and cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.
- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via intraperitoneal injection .
Conclusions:
di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not clastogenic or aneugenic in the bone marrow micronucleus test of male and female mice up to a dose of 250 mg/kg body weight and 125 mg/kg body weight, respectively
Executive summary:

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test item treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database.

 

Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genotoxicity by di-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

Bacterial mutagenicity

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was tested in theSalmonella typhimuriumreverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium(TA1535, TA1537, TA98 and TA100) and in theEscherichia colireverse mutation assay with a tryptophan-requiring strain ofEscherichia coli(WP2uvrA). The test was performed in two independent experiments in the presence and absence of 5% and 10% S9-mix (rat liver S9-mix induced by Aroclor 1254). The study followed the most recent OECD and EU protocols and was performed under GLP.

The test substance was dissolved in ethanol. The test substance precipitated on the plates at dose levels of 1568 μg/plate and upwards. The substance was tested up to 5000 µg/plate in all strains. The bacterial background lawn was not reduced at any of the concentrations tested. Only in TA1535 cytotoxicity evidence from decrease in the number of revertants at 2800 µg/plate in absence of S9-mix. Heavy precipitation at 5000 µg/plate prohibited determination of the number of revertant colonies at this dose level.

Acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

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 di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay.

 

Genotoxicity in mammalian cells:

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was tested in two independent experiments for possible clastogenicity and aneugenicity in anin vitromicronucleus assay in cultured peripheral human lymphocytes (OECD 487) in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The study followed the most recent OECD and EU protocols and was performed under GLP.

 

The test substance was dissolved in ethanol.

Positive control chemicals, mitomycin C, colchicine and cyclophosphamide induced appropriate responses indicating that the assayed worked properly.

Testing was done up to 52 µg/ml, based upon occurrence of precipitation. At 17 µg/ml no precipitation was seen in the 3 hr exposure experiments, but at all dose levels from 20 µg/ml precipitation was observed (“which does not interfere with the scoring”). No (considerable) cytotoxicity was observed at any evaluated dose levels.

Increased micronuclei levels were seen at:

- 3hr:

Without S9: exp.1. increase in binucleated cells above hist. control at 52 µg/ml; increase is significant in Cochran Armitage trend test; exp. 2: negative.

With S9: exp.1: a similar increase was seen as without S9 but was not significant; However, in exp.2: a positive, above hist.control, increase was observed at 40 and 52 µg/ml.

A positive increase in mononucleated cells only occurred at highest dose level in one of the duplicates of exp. 1

- 24hr:

No increases.

Conclusion: Although statistically significant increases were observed in the first cytogenetic assay and in the additional experiment and the number of mono- and binucleated cells with micronuclei was above the 95% control limits of the distribution of the historical negative control data, none of the increases observed in the first cytogenetic assay could be confirmed in the additional experiment. Visa versa, the increases observed in the additional experiment were not observed in the first cytogenetic assay. The report therefore concluded that the results of this study areequivocal.

Since each increase in the number of mono- and binucleated cells with micronuclei was only observed once, the biological relevance of these increases are doubtful and the results are considered to be questionable under the experimental conditions described in the report.

Levels of increased number of cells with micronuclei above lowest significant level, were only seen at the tested concentrations that also resulted to precipitation. There is no difference between with or without S9 (in exp.1); The slight increase seen in the 3 hour experiments was not seen in the 24 hr experiment, but there is only one result available in the relevant concentration range showing precipitation.

 

With the knowledge that mw is high (close to 700) and the water solubility of this substance is extremely low (< 30 µg/L; Estimations at 25°C from WSKOW v1.42 = 5.777e-012 mg/L; WATERNT  v1.01 = 6.9329e-007 mg/L) and visible precipitations was observed from 20 µg/ml, do lead to the concern that the observed increase in micronuclei is not physiologically relevant, and possible reflect an interaction of precipitation in the evaluation.To that should be considered that OECD 478 states “Even if cytotoxicity occurs above the lowest insoluble concentration, it is advisable to test at only one concentration inducing turbidity or with visible precipitate because artifactual effects may result from the precipitate.

Considering the presence of visual precipitation and the only marginal increase observed in only one of the duplicate tests, the applicant evaluates the outcome as negative rather than equivocal.

 

Mutagenicity in mammalian cells:

The mutagenic activity of di-C16-C18 (evennumbered) alkyl tripropylenetetramine was evaluated in anin vitromammalian cell gene mutation test with L5178Y mouse lymphoma cells on the

induction of forward mutations at the thymidine-kinase locus (TK-locus). The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

In the first experiment, the test item was tested up to concentrations of 52 µg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was 20% in the absence of S9-mix. No toxicity was observed at this dose level in the presence of S9-mix. After 3 hours treatment, the test item precipitated in the culture medium at the test item concentration 52 µg/ml.

In the second experiment, the test item was tested up to concentrations of 17 µg/ml in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. After 24 hours treatment, the test item precipitated in the culture medium at the test item concentration 17 µg/ml.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. Also in the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

It is concluded that di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the mouse lymphoma L5178Y test system

 

In vivo Micronucleus Test in Bone Marrow:

di-C16-C18 (evennumbered) alkyl tripropylenetetramine was tested in the bone marrow micronucleus test of male and female mice up to a dose of 250 mg/kg body weight and 125 mg/kg body weight, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test item treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database.

Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

Justification for classification or non-classification

As di-C16-C18 (evennumbered) alkyl tripropylenetetramine shows no evidence for genotoxicity, no classification for genotoxic effects is required.