Registration Dossier

Administrative data

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

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:
2009 -10-20 till 2009-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
other: TA 1535, TA 1537, TA 98, TA 100, 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 ± 2R

1 ± 1M R

6 ± 1M R

27 ± 6M R

0 ± 0M R

AND

33 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

ALKOXYPROPAN

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

DIAMINEACETATE

333 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

1000 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

2500 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

5000 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P 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 ± 3M R

138 ± 18

219 ± 15R

ALKOXYPROPAN

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

DIAMINEACETATE

333 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

1000 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

2500 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

5000 µg

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P 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 ± 23R

3 µg

14 ± 2

8 ± 3

25 ± 5

113 ± 8

56 ± 10M R

10 µg

6 ± 2R

0 ± 1M R

5 ± 3M R

29 ± 6M R

0 ± 0M R

33 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M 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 ± 11R

33 µg

13 ± 3

7 ± 1M R

14 ± 4M R

106 ± 11

229 ± 12M R

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

333 µg

0 ± 0M R P

0 ± 0P M R

0 ± 0P M R

0 ± 0P M R

0 ± 0P 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 ± 4R

9 ± 3R

25 ± 5R

78 ± 9R

231 ± 34R

10 µg

1 ± 1M R

0 ± 0M R

3 ± 1M R

14 ± 5M R

0 ± 0M R

33 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M 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 ± 4R

11 ± 1R

31 ± 3R

120 ± 10R

283 ± 15R

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M 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.

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:
other: According to OECD guideline 473
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 control1

0.0

100.0

0.5

0.5

0.0

 

 

Positive control2

0.0

66.6

8.5

8.5S

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 control1

0.0

100.0

0.5

0.0

0.0

 

 

Positive control3

0.0

44.1

14.0

13.0S

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 control1

0.0

100.0

2.0

1.5

0.0

 

 

Positive control4

0.0

75.8

13.0

12.5S

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 control1

0.0

100.0

1.5

1.5

0.0

 

 

Positive control5

0.2

54.4

11.0

10.5S

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.

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:
other: according to OECD 476
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
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. certificate)
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 106cells factor efficiency I % of 106cells 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.