Amines, N-(C18-unsatd. alkyl)trimethylenedi-, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The overall evaluation concludes that microbiological mutagenicity and mammalian mutagenicity studies show clear negative results, whereas the results from in vitro micronucleus assays in human lymphocytes are reported as equivocal.

Link to relevant study records

Reference

Reference 1

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:

20-22 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent Guideline study under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

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:

Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 & WP2uvrA-
experiment 1 and 2: All tester strains in triplicate:
without S9-mix: 0.15,0.5, 1.5, 5, 15 and 50 µg/plate
with S9-mix: 0.5, 1.5, 5, 15, 50 and 150 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

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.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett’s method of linear regression

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 applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 50 µg/plate.
A powdery precipitate was observed at 5000 ug/plate., this did not prevent the scoring of revertant colonies.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Introduction.The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 “Reverse Mutation Study”, Method B 14 of Commission Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

 

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA- were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate for all tester strains without S9-mix and 0.5 to 150 µg/plate for strains with S9-mix. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were included to allow for test material induced toxicity and to ensure that there were a minimum of four non-toxic doses plated out.

 

Results.The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 50 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied slightly between experiments depending on strain type and the presence or absence of S9-mix. The test material was, therefore, tested up to the toxic limit. A powdery precipitate was observed at 5000µg/plate, this did not prevent the scoring of revertant colonies. This observation was only made in the preliminary toxicity test because of the excessive toxicity exhibited by the test material.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

05-Sep-2011 to 28-Nov-2011

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- 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 1:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 2500 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 1000 and 2500 µg/ml
Dose range finding test 2:
Without S9-mix, 3 and 24 hours treatment: 0.01, 0.1, 1, 3, 10 and 33 µg/mL

Experiment 1:
Without S9-mix, 3 hours treatment: 0.01, 1, 1.5, 2, 3, 3.5, 4 and 4.5 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.1, 1, 5, 10, 15, 20 and 25 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.3, 0.6, 1, 1.5 and 2 µg/mL
With S9-mix, 3 hours treatment: 0.1, 10, 15, 17.5, 25, 27.5, 32.5 and 35 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and 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:

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: 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:

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.

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
Solvent control: 7.4
2500 µg/ml: 7.6
- Effects of osmolality: No
Solvent control: 0.368 mOsm/kg
2500 µg/ml: 0.326 mOsm/kg

- Precipitation: No precipitation was observed up to and including the top dose of 2500 µg/mL

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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 51 and 80% 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 89 and 60% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles.
Tris(hydroxyethyl) C12-18 alkyl diaminopropane is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

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, Tris(hydroxyethyl) C12-18 alkyl diaminopropane 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, Tris(hydroxyethyl) C12-18 alkyl diaminopropane 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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

11-Oct-2011 to 20-Mar-2012

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:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 0.3, 1, 3, 10 and 33 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 0.3, 1, 3, 10 and 33 µg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 3, 10 and 20 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 3, 10, 20 and 22 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in ethanol. 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 was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

after a short exposure period of 3 hr

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

other: equivocal by lab

Remarks:

after exposure period of 24 hr one single intermediate concentration showed a statistically significant increase in number of MN, whereas the two next higher concentrations did not (with still acceptable cytotoxicity). Should not be reagrded as positive.

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 20 µg/ml and above

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

Exp. 1A: Without metabolic activation (-S9-mix)

 

3 hours exposure time, 27 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	0	1	3		2	5
3	6	0	0	0	3		3	6
10	18	3	3	6	4		4	8
20^2)	35	2	2	4	3		4	7
0.25 MMC-C	38	0	2	2	33		38	71***
0.1 Colch	70	48	29	77***	15		13	28***

 

With metabolic activation (+S9-mix)

 

3 hours exposure time, 27 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	0	1	1	2		1	3
3	5	2	0	2	0		4	4
10	22	1	3	4	4		1	5
20^2)	25	0	3	3	4		2	6
15 CP	65	1	3	4	24		31	55***

2) Tris(hydroxyethyl) N-Tallow diaminopropane precipitated in the culture medium.

 

Exp.2: Without metabolic activation (-S9-mix)

 

24 hours exposure time, 24 hours harvest time
Concentration (µg/ml)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	2	0	2	2		1	3
3	-1	2	0	2	2		2	4
10	-3	3	1	4	5		10	15**
20^10)	10	1^2)	10^3)	11	3		1	4
22^10)	40	0^4)	0^5)	0	0^6)		0^7)	0
0.15 MMC-C	39	1	3	4	31		43	74***
0.05 Colch	92	49	94	143***	4^8)		4^9)	8

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

1) Duplicate cultures are indicated by A and B.                          

10) Tris(hydroxyethyl) N-Tallow diaminopropane precipitated in the culture medium.

Conclusions:

It is concluded that this test is valid and that Tris(hydroxyethyl) N-Tallow diaminopropane is equivocal in the formation of micronuclei in human lymphocytes in the absence of S9 metabolic activation at a prolonged exposure period. This is based on a single statistical positive MN after 24-hour exposure without S9-mix while the next two higher concentrations showed no increase of MN while showing no extreme cytotoxicity (10% resp. 40%). In view of lack of dose-response relation this single statistically significant value does not lead to the conclusion of a positive result. Furthermore, Tris(hydroxyethyl) N-Tallow diaminopropane was not clastogenic or aneugenic in human lymphocytes after 3 hours of exposure both in the absence and presence of S9-mix.

Executive summary:

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, Tris(hydroxyethyl) N-Tallow diaminopropane did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix.

In the second cytogenetic assay with a 24 hours continuous exposure time, Tris(hydroxyethyl) N-Tallow diaminopropane induced a statistically significant increase in the number of binucleated cells with micronuclei at an intermediate concentration of 10 μg/ml. Although this increase is not dose dependent, the number of binucleated cells with micronuclei is above the historical control data range.

Quality control of these slides showed an increase in the number of binucleated cells with micronuclei just above the historical control data range (6 binucleated cells with micronuclei per 1000 cells in each replicate culture) at a concentration of 10 μg/ml. These results indicate that Tris(hydroxyethyl) N-Tallow diaminopropane is equivocal in the in vitro micronucleus study.

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

The mode of action of for follows from its structure, consisting of an apolar fatty acid chain and a polar part of an ethoxylated amine. In physiological circumstances the nitrogens can become positively charged, resulting to a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. At pH below 7.2 more than 99.9% of the substance has at least one positively charged nitrogen. 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.

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

Evaluation of genetic toxicity of Tris (2-hydroxyethyl) oleyl diaminopropane (recently redefined as Amines, N-(C18 unsaturated, alkyl) trimethylenedi-, ethoxylated (NLP), CAS 1268344-02-0), also referred to here as Oelyl-diamine3EO:

For this endpoint partial read-across is applied to Coco-diamine3EO (Tris (2-hydroxyethyl) Cocoalkyl diaminopropane, CAS 90367-21-8). Data from Coco-diamine3EO is considered to represent a worst case situation for Oelyl-diamine3EO, as both substances have the same molecular structure, thus showing the exact same basic chemical reactivity, but due to its overall smaller alkyl chain length, its solubility and potential to pass cell-membranes is expected to be higher than that of Oleyl-diamine3EO. Therefore, any inherent genotoxic hazards are more likely to be expressed when testing with Coco-diamine3EO than with Oleyl-diamine3EO.  

 

The following studies are available Oleyl-diamine3EO and Coco-diamine3EO for the assessment of genetic toxicity. Al tests are performed to current OECD/EU protocols and in compliance to GLP.

 

Oleyl-diamine3EO was tested in Salmonella typhimuriums trains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. All controls showed adequate responses. The test material was tested up to the toxic limit. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Coco-diamine3EO was also tested in this Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat), and 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.

 

Coco-diamine3EO was assessed for its potential to induce gene mutations at the Thymidine kinase (TK) locus using mouse lymphoma L5178Y. The test was performed in two independent experiments in the absence and presence of S9-mix. In both the presence and absence of S9-mix, Coco-diamine3EO did not induce a significant increase in the mutation frequency.

 

Coco-diamine3EO was further evaluated for possible clastogenicity and aneugenicity in an in vitro micronucleus assay in cultured peripheral human lymphocytes (with independent repeat) in the presence and absence of a metabolic activation system. The study was performed according to OECD 487 and in compliance to GLP. The results of the positive and negative controls indicate that the conditions of the test were adequate.

The report concludes that that Coco-diamine3EO induces the formation of micronuclei in human lymphocytes in the absence of S9 metabolic activation after a short exposure period.

This is based on only one statistical positive MN in the 3-hour exposure experiment without S9 at 25 µg/mL, showing no excessive cytotoxicity (33%). However, the next higher concentration of 30 µg/mL showed no increase of MN while still showing no extreme cytotoxicity (38%). In view of lack of dose-response relation this single statistically significant value should not lead to the conclusion of a positive result. Besides, after 24-hour exposure without S9-mix, even the highest concentration evaluated of 15 µg/mL before toxicity becomes too high (from 20 µg/mL not enough cells are available for CBPI evaluation) did not result in an increase of MN. This is suggestive that in the short time exposure this concentration would also not lead to an increase of MN, and suggest that the increase observed at 25 µg/mL is just accidental.

 

A similar micronucleus assay was done on Tallow-diamine3EO under the same test conditions. Contrary to the Coco-diamine3EO, the results of this study show no increase in number of MN after a short exposure period. However, in this study now a single statistically significant increase in the number of binucleated cells with micronuclei was observed at an intermediate concentration of 10 µg/mL at prolonged exposure duration only.However, the next higher concentrations of 20 and 22 µg/mL showed no increase of MN (4 resp. 0) while both showing no extreme cytotoxicity and thus still acceptable for scoring (10% resp. 40%).Although this increase seems accidental and is not dose dependent, the number of binucleated cells with micronuclei is above the historical control data range. The report therefore concludes that Tallow-diamine3EO is equivocal in thein vitromicronucleus study.

 

When comparing the MN studies on Coco-diamine3EO closely to the Tallow-diamine3EO, the following observations can be made:

- Both studies basically showed the same picture: A small increase in micronuclei can be observed at toxic dose levels without needing metabolic activation.

- The coco-derivative of ethoxylated diamine is somewhat better water soluble, and no precipitation has been observed.

- Contrary to the tallow, shows the coco substance a somewhat higher toxicity with longer exposure duration.

- Similar to tallow, there are no differences between assays with or without metabolic activation with respect to cytotoxicity. (Which corresponds with the notion that these substances do not undergo important phase-one metabolism). Also with S9 it looks like some increase of micronuclei is visible for Coco-diamine3EO, but only without S9 it reaches a statically significance at one concentration. This could be because 30 and 40µg/ml were possibly too toxic (also lack of mononucleated cells to score and at 40 even also of binucleated cells), and the 25 µg/ml that showed the increase without metabolic activation was not evaluated in the presence of S9.

- The small increase of micronuclei at toxic levels is similar as for tallow, and again not systematically observed. In this case in the short exposure period, but not in the long exposure assay as for Tallow.

- The effects are only seen in combination of toxicity. In the slides evaluated as positive, there were not enough mononucleated cells available for scoring, indicating an extreme loss of cells. If this cell loss was due to toxicity, this could be indication for excessive toxicity (> 50% cell death is generally considered as excessive)

- For both Coco and Tallow-diamine3EO there is no dose-relationship observed. In case of absence of dose relation, a substance can still be considered positive in case the increase is biologically relevant. This should be interpreted for the evaluation where only the highest dose which is not too toxic shows an really significant increase which is not just passing the level of statistical significance or the limit seen in historical controls. In both cases it can be questioned whether this criteria of biological significance is really reached: response are marginal, and not consistent, and at levels that show (possible too high) toxicity, and not showing clear dose-response relation.

 

In conclusion, both studies evaluating the possible clastogenicity and aneugenicity properties of Coco-diamine3EO and Tallow-diamine3EO in an in vitro micronucleus assay in cultured peripheral human lymphocytes show consistent results that can, on overall, be characterised as inconsistent, possibly weakly positive responses at cytotoxic levels.

 

The overall evaluation concludes that microbiological mutagenicity and mammalian mutagenicity studies show negative results, whereas the results from in vitro micronucleus assays in human lymphocytes can only be described as equivocal.

These obtained results are consistent, and do not lead to a need for classification.

Further studies are not expected to improve upon the available data, and are therefore not proposed.

Justification for classification or non-classification

The overall evaluation concludes that microbiological mutagenicity and mammalian mutagenicity studies show negative results, whereas the results from in vitro micronucleus assays in human lymphocytes can only be described as equivocal. The overall conclusion is therefore 'inconclusive'.

These results obtained results are consistent, and do not lead to a need for classification for genotoxicity.

Further studies are not expected to improve upon the available data, and are therefore not proposed.