1,3-Propanediamine, N-[3-((C11-14, C13-rich)oxy)propyl]- branched acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Etherdiamine C13i/acetate is not mutagenic in the Salmonella typhimurium reverse mutation assay, is not clastogenic in human lymphocytes, and not mutagenic in the HPRT mutation test with Chinese hamster V79 cells.

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:

2009 -10-20 till 2009-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Etherdiamine C13i/acetate: Standard Commercial grade

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment I
without S9 mix: 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 µg/plate
with S9 mix: 0.03; 0.1; 0.3; 1; 3; 10; 33; 100; and 333 µg/plate
Experiment II
without S9 mix: 0.01; 0,03; 0.1; 0.3; 1; 3; 10; and 33 µg/plate
with S9 mix: 0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: better than others

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar: plate incorporation and preincubation


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, andTA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility:
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 1000 µg/plate up to 5000 µg/plate in the pre-experiment and at 333 µg/plate in experiment I. Precipitation of the test item on the incubated agar plates was observed from 333 µg/plate up to 5000 µg/plate in the pre-experiment and at 333 µg/plate in experiment I. The undissolved particles of the test item had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):
Strain Pre-Experiment Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 10 - 5000 100 - 5000 10 - 100 100 - 333 3 - 33 33 - 100
TA 1537 10 - 5000 100 - 5000 10 - 100 33 - 333 3 - 33 33 - 100
TA 98 10 - 5000 33 - 5000 10 - 100 33 - 333 3 - 33 33 - 100
TA 100 10 - 5000 100 - 5000 10 - 100 100 - 333 3 - 33 33 - 100
TA 102 3 - 5000 33 - 5000 1 - 100 10 - 333 3 - 33 33 - 100
Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Pre-Experiment Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 33 - 5000 100 - 5000 33 - 100 100 - 333 10 - 33 100
TA 1537 10 - 5000 100 - 5000 10 - 100 33 - 333 10 - 33 100
TA 98 10 - 5000 100 - 5000 10 – 100 33 - 333 10 - 33 100
TA 100 10 - 5000 100 - 5000 10 – 100 100 - 333 10 - 33 100
TA 102 3 - 5000 33 - 5000 3 - 100 33 - 333 10 - 33 100

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Summary of Results Pre-Experiment

Study Name: 1291001	Study Code: Harlan CCR 1291001
Experiment: 1291001 VV Plate	Date Plated: 20/10/2009
Assay Conditions:	Date Counted: 23/10/2009

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			17 ± 4	17 ± 2	35 ± 1	153 ± 15	458 ± 6
	Untreated			13 ± 1	12 ± 6	39 ± 11	163 ± 6	452 ± 11
	ALKOXYPROPAN	3 µg		18 ± 5	12 ± 3	31 ± 6	128 ± 11	^N R
	DIAMINE	10 µg		10 ± 2^R	1 ± 1^M R	6 ± 1^M R	27 ± 6^M R	0 ± 0^M R
	AND	33 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	ALKOXYPROPAN	100 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	DIAMINEACETATE	333 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		1000 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		2500 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		5000 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
	NaN3	10 µg		1928 ± 130			2129 ± 120
	4-NOPD	10 µg				356 ± 6
	4-NOPD	50 µg			105 ± 6
	MMS	3.0 µL						3492 ± 696
With Activation	Deionised water			18 ± 5	18 ± 5	47 ± 6	173 ± 10	593 ± 39
	Untreated			17 ± 3	16 ± 4	40 ± 6	177 ± 2	587 ± 40
	ALKOXYPROPAN	3 µg		16 ± 4	18 ± 4	44 ± 6	185 ± 16	565 ± 50
	DIAMINE	10 µg		15 ± 1	19 ± 4	40 ± 10	178 ± 14	565 ± 11
	AND	33 µg		16 ± 3	13 ± 2	22 ± 3^M R	138 ± 18	219 ± 15^R
	ALKOXYPROPAN	100 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	DIAMINEACETATE	333 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		1000 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		2500 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		5000 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
	2-AA	2.5 µg		445 ± 31	291 ± 3	2618 ± 50	2688 ± 82
	2-AA	10.0 µg						2314 ± 55

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine	M R P N	Manual count Reduced background growth Precipitate Analysis not possible

Summary of Results Experiment I

Study Name: 1291001	Study Code: Harlan CCR 1291001
Experiment: 1291001 HV1 Plate	Date Plated: 03/11/2009
Assay Conditions:	Date Counted: 06/11/2009

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			11 ± 4	11 ± 3	25 ± 7	124 ± 8	371 ± 13
	Untreated			13 ± 2	13 ± 3	23 ± 4	128 ± 6	341 ± 23
	ALKOXYPROPAN	0.01 µg		13 ± 3	8 ± 3	29 ± 1	131 ± 13	408 ± 21
	DIAMINE	0.03 µg		13 ± 2	9 ± 5	27 ± 6	128 ± 7	359 ± 13
	AND	0.1 µg		12 ± 2	12 ± 3	25 ± 3	124 ± 4	375 ± 10
	ALKOXYPROPAN	0.3 µg		13 ± 3	9 ± 1	28 ± 4	119 ± 12	388 ± 10
	DIAMINEACETATE	1 µg		9 ± 1	9 ± 3	25 ± 3	122 ± 3	306 ± 23^R
		3 µg		14 ± 2	8 ± 3	25 ± 5	113 ± 8	56 ± 10^M R
		10 µg		6 ± 2^R	0 ± 1^M R	5 ± 3^M R	29 ± 6^M R	0 ± 0^M R
		33 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
		100 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	NaN3	10 µg		1812 ± 65			1955 ± 42
	4-NOPD	10 µg				261 ± 4
	4-NOPD	50 µg			68 ± 5
	MMS	3.0 µL						3088 ± 84
With Activation	Deionised water			15 ± 2	17 ± 4	37 ± 5	140 ± 12	551 ± 34
	Untreated			16 ± 2	16 ± 6	35 ± 2	136 ± 17	507 ± 22
	ALKOXYPROPAN	0.03 µg		15 ± 1	15 ± 1	33 ± 7	145 ± 6	577 ± 5
	DIAMINE	0.1 µg		12 ± 3	11 ± 2	34 ± 1	128 ± 17	545 ± 24
	AND	0.3 µg		15 ± 4	13 ± 5	42 ± 5	135 ± 6	579 ± 34
	ALKOXYPROPAN	1 µg		15 ± 4	16 ± 4	37 ± 3	139 ± 8	598 ± 20
	DIAMINEACETATE	3 µg		15 ± 1	15 ± 1	33 ± 8	164 ± 7	569 ± 16
		10 µg		11 ± 3	16 ± 2	38 ± 4	148 ± 12	426 ± 11^R
		33 µg		13 ± 3	7 ± 1^M R	14 ± 4^M R	106 ± 11	229 ± 12^M R
		100 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
		333 µg		0 ± 0^M R P	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
	2-AA	2.5 µg		313 ± 24	240 ± 18	1766 ± 444	2560 ± 60
	2-AA	10.0 µg						2133 ± 195

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine	M R P	Manual count Reduced background growth Precipitate

Summary of Results Experiment II

Study Name: 1291001	Study Code: Harlan CCR 1291001
Experiment: 1291001 HV2 pre	Date Plated: 16/11/2009
Assay Conditions:	Date Counted: 19/11/2009

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			15 ± 6	14 ± 2	32 ± 5	129 ± 17	407 ± 26
	Untreated			12 ± 4	10 ± 2	24 ± 3	139 ± 19	396 ± 16
	ALKOXYPROPAN	0.01 µg		13 ± 2	11 ± 2	29 ± 4	139 ± 18	452 ± 15
	DIAMINE	0.03 µg		16 ± 2	12 ± 2	26 ± 8	127 ± 17	393 ± 28
	AND	0.1 µg		16 ± 2	8 ± 3	35 ± 6	129 ± 14	454 ± 18
	ALKOXYPROPAN	0.3 µg		15 ± 3	13 ± 1	26 ± 5	134 ± 11	420 ± 12
	DIAMINEACETATE	1 µg		11 ± 3	8 ± 2	29 ± 5	120 ± 15	374 ± 3
		3 µg		13 ± 4^R	9 ± 3^R	25 ± 5^R	78 ± 9^R	231 ± 34^R
		10 µg		1 ± 1^M R	0 ± 0^M R	3 ± 1^M R	14 ± 5^M R	0 ± 0^M R
		33 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	NaN3	10 µg		1804 ± 30			1791 ± 115
	4-NOPD	10 µg				349 ± 53
	4-NOPD	50 µg			71 ± 6
	MMS	3.0 µL						3068 ± 246
With Activation	Deionised water			21 ± 3	15 ± 2	35 ± 4	170 ± 8	626 ± 29
	Untreated			16 ± 6	14 ± 1	45 ± 6	168 ± 31	570 ± 51
	ALKOXYPROPAN	0.03 µg		18 ± 4	16 ± 3	39 ± 7	153 ± 11	658 ± 26
	DIAMINE	0.1 µg		19 ± 4	15 ± 1	35 ± 3	154 ± 18	637 ± 16
	AND	0.3 µg		23 ± 3	13 ± 2	42 ± 11	156 ± 7	646 ± 18
With Activation	ALKOXYPROPAN	1 µg		22 ± 2	22 ± 3	40 ± 9	162 ± 11	586 ± 57
	DIAMINEACETATE	3 µg		25 ± 2	13 ± 6	34 ± 7	145 ± 6	570 ± 27
		10 µg		20 ± 7	15 ± 3	42 ± 7	150 ± 7	498 ± 51
		33 µg		15 ± 4^R	11 ± 1^R	31 ± 3^R	120 ± 10^R	283 ± 15^R
		100 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	2-AA	2.5 µg		348 ± 21	292 ± 29	2258 ± 171	2448 ± 127
	2-AA	10.0 µg						2907 ± 730

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine	R M	Reduced background growth Manual count

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of Alkoxypropandiamine and Alkoxypropandiamineacetate to induce gene muta­tions according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment:                        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment I
without S9 mix:                        0.01; 0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 µg/plate
with S9 mix:                        0.03; 0.1; 0.3; 1; 3; 10; 33; 100; and 333 µg/plate

Experiment II
without S9 mix:                        0.01; 0,03; 0.1; 0.3; 1; 3; 10; and 33 µg/plate
with S9 mix:                        0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 µg/plate

The plates incubated with the test item showed reduced back­ground growth with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Alkoxypropandiamine and Alkoxypropandiamineacetate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-05 till 2009-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Etherdiamine C13i/acetate: Standard Commercial grade

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 1.3, 2.3, 4.0, 7.0 12.2, 21.3, 37.3, 65.3, 114.3, 200.0 µg/mL
Experiment II: 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0 µg/mL

Without metabolic activation:
Experiment I: 1.3, 2.3, 4.0, 7.0, 12.2, 21.3, 37.3, 65.3, 114.3, 200.0 µg/mL
Experiment II: 0.04, 0.06, 0.1, 0.2, 0.4, 0.6, 1.1, 1.9, 3.3, 5.7, 10.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Alkoxypropandiamine and Alkoxypropandiamine acetate, dissolved in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphase plates per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 4125.0 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the molecular weight and the purity (90.7 %) of the test item.
Precipitation of the test item in the culture medium was only observed in Experiment I at 37.3 µg/mL and above in the presence of S9 mix. No relevant increase in the osmolarity or pH value was observed (Exp. I: solvent control: 277 mOsm, pH 7.3 versus 280 mOsm and pH 7.3 at 200.0 µg/mL; Exp. II: solvent control: 276 mOsm, pH 7.7 versus 274 mOsm and pH 7.7 at 10.0 µg/mL).
Clearly reduced mitotic indices were observed in Experiment I in the absence of S9 mix (35.5 % of control) and in Experiment II in the presence of S9 mix (45.3 % of control). In Experiment I in the presence of S9 mix and in Experiment II in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, the mitotic indices were greatly reduced to approx. 60 % of control at the highest evaluated concentrations.
In both experiments in the absence and presence of S9 mix no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical solvent control data.
Table 5 and 8, pages 24 and 27, show the occurrence of polyploid metaphases. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 - 0.2 %) as compared to the rate of the solvent controls (0.0 %).
In both experiments, either EMS (660.0 or 825.0 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Table 1- Doses applied in the chromosome aberration assay with Alkoxypropandiamine and Alkoxypropandiamine acetate

Exp.	Prep. interval	Exposure period	Concentrations in µg/ml
Without S9 mix
I	22 hrs	4 hrs		1.3	2.3	4.0	7.0	12.2	21.3	37.3	65.3	114.3	200.0
II	22 hrs	22 hrs	0.04	0.06	0.1	0.2	0.4	0.6	1.1	1.9	3.3	5.7	10.0
With S9 mix
I	22 hrs	4 hrs		1.3	2.3	4.0	7.0	12.2	21.3	37.3p	65.3p	114.3p	200.0p
II	22 hrs	4 hrs		1.0	2.0	3.0	4.0	5.0	6.0	7.0	8.0	10.0	12.0

Evaluated experimental points are shown in bold characters

P Precipitation was observed at the end of treatment

Table 2 - Summary of results of the chromosomal aberration study with Alkoxypropandiamine and Alkoxypropandiamine acetate

Exp.	Preparation	Test item	Polyploid	Mitotic indices	Aberrant cells
	interval	concentration	cells	in %	in %
		in µg/mL	in %	of control	incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	0.0	100.0	0.5	0.5	0.0
		Positive control^2	0.0	66.6	8.5	8.5^S	1.0
		1.3	0.2	91.6	3.0	2.5	1.0
		2.3	0.0	67.8	1.5	1.5	0.5
		4.0	0.0	35.5	2.0	1.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	0.0	100.0	0.5	0.0	0.0
		Positive control^3	0.0	44.1	14.0	13.0^S	3.0
		1.9	0.0	87.3	1.0	1.0	0.0
		3.3	0.0	92.3	2.0	1.5	0.0
		5.7	0.0	62.5	2.0	1.0	0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	0.0	100.0	2.0	1.5	0.0
		Positive control^4	0.0	75.8	13.0	12.5^S	3.0
		2.3	0.2	110.2	3.0	2.0	0.0
		4.0	0.2	102.7	0.5	0.5	0.0
		7.0	0.2	62.5	2.0	2.0	0.0
II	22 hrs	Solvent control^1	0.0	100.0	1.5	1.5	0.0
		Positive control^5	0.2	54.4	11.0	10.5^S	1.0
		6.0	0.2	67.6	1.0	1.0	0.0
		8.0	0.0	64.2	2.0	2.0	0.0
		10.0	0.0	45.3	1.0	1.0	0.0

*  Including cells carrying exchanges

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Deionised water 10.0 % (v/v)

²   EMS 825.0 µg/mL

³   EMS 660.0 µg/mL

⁴   CPA     7.5 µg/mL

⁵   CPA   15.0 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, Alkoxypropandiamine and Alkoxypropandiamine acetate is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentrations.

Executive summary:

The test item Alkoxypropandiamine and Alkoxypropandiamine acetate, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in the pre-test on toxicity (4125.0 µg/mL of the test item) was chosen with regard to the molecular weight and the purity (90.7 %) of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The chosen treatment concentrations and the rationale for the dose selection are reported in Table1. The evaluated experimental points and the results are summarised in Table2.

In Experiment I, in the absence of S9 mix and in Experiment II, in the presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In Experiment I, in the presence of S9 mix and in Experiment II, in the absence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, the mitotic index was greatly reduced to approx. 60 % of control at the highest evaluated concentrations.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Etherdiamine C13i/acetate: Standard Commercial grade

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation:0.063; 0.13; 0.25; 0.50; 0.75; and 1.00 µg/mL (4 hours treatment)
with metabolic activation: 0.5; 1.0; 2.0; 4.0; 6.0; and 8.0 µg/mL (4 hours treatment)
Experiment II:
without metabolic activation: 0.031; 0.063; 0.130; 0.25, 0.5; and 0.75 µg/mL (24 hours treatment)
with metabolic activation: 1.0; 2.0; 4.0; 6.0; 7.0; and 8.0 µg/mL (4 hours treatment)
The cultures at 0.5 µg/mL (printed in bold) in the first experiment with metabolic activation and at 0.031 µg/mL in the second experiment without metabolic activation were not continued, since a minimum of only four analysable concentrations is required by the guidelines. The cultures at 0.75 and 1.0 µg/mL in the first experiment without metabolic activation and at 8.0 µg/mL in the second experiment with metabolic activation were not continued due to exceedingly severe cytotoxic effects.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

Linear regression analysis (least squares) .

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic: with S9: from 6.0 µg/mL; without S9 from 0.25 µg/mL (exp. 1) and 0.75 µg/mL (exp.2)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Water solubility: Not indicated by the sponsor
- Precipitation:
Precipitation occurred in the pre-experiment at the end of treatment at 32.8 µg/mL and above with metabolic activation and at 525 µg/mL
without metabolic activation and 4h treatment. After 24h treatment without metabolic activation precipitation was noted at 262.5 µg/mL and above.
In the main experiments no precipitation was observed.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiments test item concentrations between 0.5 and 4200 µg/mL (approximately 10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Relevant to severe cytotoxic effects were noted at 0.5 µg/mL and above following 4h and 24h treatment without metabolic activation and at 8.0 µg/mL and above following 4h treatment with metabolic activation. Precipitation at the end of treatment was observed at 32.8 µg/mL and above with metabolic activation and at 525 µg/mL without metabolic activation and 4h treatment. After 24h treatment without metabolic activation precipitation was noted at 262.5 µg/mL and above.
There was no relevant shift of the osmolarity of the medium or the pH even at the maximum concentration without metabolic activation.
Based on the results of the pre-experiments the concentration range of the main experiments was selected. The individual concentrations were generally spaced by a factor of 2.0. At high concentrations a narrower spacing was used to cover the cytotoxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: within historical solvent control range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I below 50% were observed in the first experiment at 6.0 µg/mL and above with and at 0.25 µg/mL and above without metabolic activation. In the second experiment toxic effects as described above occurred at 6.0 µg/mL and above with and at 0.75 µg/mL without metabolic activation.

Summary Table

			relative	relative	mutant		relative	relative	mutant
	conc. µg	S9	cloning	cell density	colonies/	induction	cloning	cell density	colonies/	induction
	per mL	mix	efficiency I	% of	10^6cells	factor	efficiency I	% of	10^6cells	factor
			%	control			%	control
Column	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 h treatment			culture I				culture II
Solvent control with water		-	100.0	100.0	10.7	1.0	100.0	100.0	8.6	1.0
Positive control with EMS	150.0	-	73.6	79.8	162.3	15.2	34.8	87.9	83.0	9.7
Test item	0.063	-	109.4	87.7	11.4	1.1	92.7	96.3	13.8	1.6
Test item	0.13	-	103.5	86.5	6.9	0.7	94.2	102.6	6.2	0.7
Test item	0.25	-	32.9	91.0	14.3	1.3	60.2	98.5	13.1	1.5
Test item	0.50	-	0.0	21.7	16.6	1.6	0.7	82.4	5.8	0.7
Test item	0.75	-	0.0	culture was not continued^#			0.0	culture was not continued^#
Test item	1.00	-	0.0	culture was not continued^#			0.0	culture was not continued^#
Solvent control with water		+	100.0	100.0	15.9	1.0	100.0	100.0	5.6	1.0
Positive control with DMBA	1.1	+	71.7	79.0	344.9	21.7	74.4	77.0	264.9	47.2
Test item	0.5	+	82.5	culture was not continued^##			86.9	culture was not continued^##
Test item	1.0	+	72.1	99.9	11.9	0.7	91.3	115.3	13.5	2.4
Test item	2.0	+	81.8	99.3	9.2	0.6	85.5	109.6	5.9	1.1
Test item	4.0	+	76.1	113.9	7.9	0.5	81.7	104.3	6.2	1.1
Test item	6.0	+	29.1	85.2	10.2	0.6	32.3	68.0	6.7	1.2
Test item	8.0	+	0.0	14.4	5.7	0.4	0.3	17.1	20.0	3.6
Experiment II / 24 h treatment			culture I				culture II
Solvent control with water		-	100.0	100.0	23.2	1.0	100.0	100.0	14.8	1.0
Positive control with EMS	150.0	-	87.4	140.7	371.1	16.0	86.8	87.4	273.0	18.4
Test item	0.031	-	101.4	culture was not continued^##			92.5	culture was not continued^##
Test item	0.063	-	98.8	125.9	12.3	0.5	96.0	83.7	17.9	1.2
Test item	0.130	-	99.3	120.3	33.3	1.4	97.4	83.5	3.1	0.2
Test item	0.250	-	100.4	126.9	16.3	0.7	96.5	79.5	15.4	1.0
Test item	0.500	-	66.0	124.9	26.5	1.1	60.0	65.8	13.6	0.9
Test item	0.750	-	29.4	92.3	18.7	0.8	22.1	65.0	23.1	1.6
Experiment II / 4 h treatment
Solvent control with water		+	100.0	100.0	16.8	1.0	100.0	100.0	14.8	1.0
Positive control with DMBA	1.1	+	75.4	67.3	635.7	37.7	80.0	65.7	587.8	39.8
Test item	1.0	+	98.0	96.7	30.2	1.8	97.0	82.8	21.0	1.4
Test item	2.0	+	94.1	82.1	22.5	1.3	94.6	84.8	9.8	0.7
Test item	4.0	+	87.0	93.7	33.7	2.0	87.3	100.8	16.4	1.1
Test item	6.0	+	31.8	72.3	31.8	1.9	40.7	90.3	15.4	1.0
Test item	7.0	+	11.0	37.0	52.5	3.1	7.3	58.6	41.6	2.8
Test item	8.0	+	0.0	culture was not continued^#			0.0	culture was not continued^#

#  culture was not continued due to exceedingly severe cytotoxic effects

##culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, a Mixture of Alkoxypropandiamine acetate (CAS 102047-27-8) and Alkoxypropandiamine (CAS 68479-04-9) is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item Mixture of Alkoxypropandiamine acetate (CAS 102047-27-8) and Alkoxypropandiamine (CAS 68479-04-9) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:          0.063; 0.13; 0.25; and 0.5 µg/mL
with S9 mix:                1.0; 2.0; 4.0; 6.0; and 8.0 µg/mL

Experiment II:

without S9 mix:           0.063; 0.13; 0.25; 0.5; and 0.75 µg/mL
with S9 mix:                 1.0; 2.0; 4.0; 6.0; and 7.0 µg/mL

Relevant cytotoxic effects indicated by a relative cloning efficiency I below 50% were observed in the first experiment at 6.0 µg/mL and above with and at 0.25 µg/mL and above without metabolic activation. In the second experiment toxic effects as described above occurred at 6.0 µg/mL and above with and at 0.75 µg/mL without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximal concentration. The induction factor of three times the corresponding solvent control was exceeded at 8.0 µg/mL in the second culture of the first experiment with metabolic activation and at 7.0 µg/mL in the first culture of the second experiment with metabolic activation. However, the induction factor of each parallel culture remained below the threshold. Only one out of four experimental parts with metabolic activation (two cultures of experiment I and two cultures of experiment II) showed a statistically significant increase of the mutation frequency (culture I of the second experiment with metabolic activation). Therefore, the increased values of the mutation frequency described above are considered irreproducible and thus biologically irrelevant toxic artefacts

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the first culture of the second experiment with metabolic activation. Another significant trend noted in the first culture of the first experiment with metabolic activation was judged as irrelevant since it was reciprocal, going down versus increasing concentrations.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.6 up to 23.2 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.1 up to 52.5 mutants per 10⁶cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genototoxicity by EtherdiamineC13i/acetateis not expected. In physiological circumstances, EtherdiamineC13i/acetatehas a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tail 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

Etherdiamine C13i/acetate has been tested for genetic toxicity in three in vitro tests to current OECD/EU protocols carried out according to GLP and with a clearly defined and described test substance.

 

Etherdiamine C13i/acetate was tested in the Salmonella typhimurium reverse mutation assay with five histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, and TA 102). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced with Phenobarbital/β-Naphthoflavone).
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.

 

Etherdiamine C13i/acetate 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.

Etherdiamine C13i/acetate 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. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

 

Etherdiamine C13i/acetate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced with Phenobarbital/β-Naphthoflavone).

In both the presence and absence of S9-mix, Etherdiamine C13i/acetate did not induce a significant increase in the mutation frequency.

Justification for classification or non-classification

Etherdiamine C13i/acetate is not mutagenic in the Salmonella typhimurium reverse mutation assay, is not clastogenic in human lymphocytes, and not mutagenic in the HPRT mutation test with Chinese hamster V79 cells.

It can therefore be concluded that Etherdiamine C13i/acetate is not genotoxic.