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

Additional information

Additional information from genetic toxicity in vitro:

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 withPhenobarbital/β-Naphthoflavone).

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


Justification for selection of genetic toxicity endpoint
For each endpoint, bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a GLP compliant study is available.

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.