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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2010 -03-22 till 2010-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N, N, N´,N´,N´´-Pentamethyl-N-tallowalkyl- 1,3- propanediammonium chloride
- Physical state: solid yellow
- Analytical purity: 94.5 %
- Lot/batch No.: S001702
- Expiration date of the lot/batch: March 2017
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: At room temperature, under nitrogen, moisture protected

Method

Species / strain
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/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment I and II without S9 mix: 0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 µg/plate
Experiment I and II with S9 mix: 0.3; 1; 3; 10; 33; 100; 250; and 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: better than others
Controls
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: iplate incorporation; 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, and TA 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.

Results and discussion

Test results
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
- Precipitation:
The test item precipitated in the overlay agar on the incubated in the pre-experiment at 2500 µg/plate and 5000 µg/plate in the absence of metabolic activation and from 1000 µg/plate up to 5000 µg/plate in the presence of metabolic activation. In Experiment I and II precipitation of the test item was observed from 100 µg/plate to 500 µg/plate in the presence of metabolic activation. 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:
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 - 500 10 - 100 100 - 500
TA 1537 10 - 5000 100 - 5000 33 - 100 250 - 500 10 - 100 100 - 500
TA 98 10 - 5000 100 - 5000 33 - 100 250 - 500 10 - 100 100 - 500
TA 100 10 - 5000 100 - 5000 10 - 100 / 10 - 100 100 - 500
TA 102 10 - 5000 100 - 5000 10 - 100 / 10 - 100 100 - 500
/ = no reduced background growth
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 333 - 5000 33 - 100 250 - 500 10 - 100 250 - 500
TA 1537 33 - 5000 333 - 5000 33 - 100 250 - 500 10 - 100 250 - 500
TA 98 33 - 5000 333 - 5000 100 250 - 500 33 - 100 250 - 500
TA 100 33 - 5000 333 - 5000 33 - 100 250 - 500 33 - 100 100 - 500
TA 102 33 - 5000 333 - 5000 33 - 100 250 - 500 10 - 100 250 - 500
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Pre-Experiment

Study Name: 1302501

Study Code: Harlan CCR 1302501

Experiment: 1302501 VV Plate

Date Plated: 22/03/2010

Assay Conditions:

Date Counted: 25/03/2010

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

DMSO

15 ± 6

14 ± 3

25 ± 5

158 ± 12

444 ± 14

Untreated

13 ± 3

19 ± 7

24 ± 5

173 ± 13

416 ± 27

N, N, N´,N´,N´´-

3 µg

10 ± 3

16 ± 1

25 ± 6

157 ± 11

364 ± 21

Pentamethyl-N-

10 µg

9 ± 3R

14 ± 4R

25 ± 5R

110 ± 7R

364 ± 39R

tallowalkyl- 1,3-

33 µg

4 ± 1M R

1 ± 2M R

1 ± 1M R

4 ± 3M R

37 ± 3M R

propanediammonium

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

chloride

333 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

1000 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

2500 µg

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

5000 µg

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

NaN3

10 µg

1734 ± 53

1887 ± 122

4-NOPD

10 µg

346 ± 17

4-NOPD

50 µg

71 ± 5

MMS

3.0 µL

2258 ± 210

With Activation

DMSO

17 ± 4

22 ± 1

31 ± 3

165 ± 20

586 ± 35

Untreated

18 ± 3

19 ± 2

43 ± 7

177 ± 15

538 ± 66

N, N, N´,N´,N´´-

3 µg

15 ± 3

23 ± 4

38 ± 8

161 ± 7

519 ± 14

Pentamethyl-N-

10 µg

15 ± 5

23 ± 5

35 ± 7

179 ± 9

567 ± 40

tallowalkyl- 1,3-

33 µg

13 ± 4

30 ± 6

41 ± 10

182 ± 3

595 ± 12

propanediammonium

100 µg

12 ± 4R

19 ± 5R

29 ± 7R

53 ± 30R

363 ± 39R

chloride

333 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

1000 µg

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

2500 µg

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

5000 µg

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

0 ± 0M R P

2-AA

2.5 µg

500 ± 45

503 ± 50

2133 ± 551

4080 ± 219

2-AA

10.0 µg

2397 ± 57

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

P

Reduced background growth

Manual count

Precipitate

  Summary of Results Experiment I

Study Name: 1302501

Study Code: Harlan CCR 1302501

Experiment: 1302501 HV1 Pre

Date Plated: 13/04/2010 / 21/04/2010*

Assay Conditions:

Date Counted: 16/04/2010 / 28/04/2010*

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

DMSO

17 ± 1

15 ± 6

35 ± 3

131 ± 9

447 ± 8

Untreated

15 ± 4

8 ± 1

33 ± 5

132 ± 9

479 ± 24

N, N, N´,N´,N´´-

0.03 µg

19 ± 4

13 ± 3

28 ± 1

135 ± 4

459 ± 44

Pentamethyl-N-

0.1 µg

16 ± 1

14 ± 4

35 ± 7

137 ± 11

437 ± 15

tallowalkyl- 1,3-

0.3 µg

17 ± 3

10 ± 5

26 ± 6

140 ± 4

427 ± 14

propanediammonium

1µg

15 ± 1

12 ± 3

30 ± 3

136 ± 7

428 ± 18

chloride

3 µg

21 ± 5

12 ± 4

28 ± 7

134 ± 7

451 ± 7

10 µg

18 ± 2R

11 ± 5

27 ± 3

99 ± 7R

390 ± 13R

33 µg

3 ± 1M R

4 ± 1M R

16 ± 3R

3 ± 2M R

16 ± 3M R

100 µg

0 ± 0M R

1 ± 2M R

1 ± 1M R

0 ± 0M R

0 ± 0M R

NaN3

10 µg

1929 ± 133

338 ± 28

2065 ± 35

4-NOPD

10 µg

4-NOPD

50 µg

82 ± 2

MMS

3.0 µL

4677 ± 818

With Activation

DMSO

22 ± 2

19 ± 7

47 ± 6

143 ± 8

563 ± 117

Untreated

16 ± 6

29 ± 0

47 ± 10

166 ± 26

555 ± 31

N, N, N´,N´,N´´-

0.3 µg

17 ± 4

22 ± 6

49 ± 10

136 ± 10

514 ± 29

Pentamethyl-N-

1 µg

19 ± 5

24 ± 2

38 ± 6

152 ± 7

540 ± 43

tallowalkyl- 1,3-

3 µg

24 ± 8

13 ± 6

43 ± 5

148 ± 9

519 ± 27

propanediammonium

10 µg

15 ± 2

21 ± 3

41 ± 8

140 ± 18

485 ± 29

chloride

33 µg

19 ± 2

21 ± 5

41 ± 9

149 ± 14

510 ± 18

100 µg

18 ± 5R P

12 ± 5P

36 ± 3P

147 ± 17P

418 ± 45P

250 µg

0 ± 1P R M

6 ± 2M R P

5 ± 4P M R

0 ± 0P M

20 ± 5P M

500 µg

0 ± 0P M R

0 ± 0M R P

0 ± 0P M R

0 ± 0P M

0 ± 0P M

2-AA

2.5 µg

550 ± 40

482 ± 5

2251 ± 25

2911 ± 77

2-AA

10.0 µg

2905 ± 190

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

P

Reduced background growth

Manual count

Precipitate

* = repeated experiment

  Summary of Results Experiment II

Study Name: 1302501

Study Code: Harlan CCR 1302501

Experiment: 1302501 HV2 Pre

Date Plated: 22/04/2010

Assay Conditions:

Date Counted: 28/04/2010

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

DMSO

16 ± 6

11 ± 2

30 ± 9

122 ± 3

310 ± 2

Untreated

15 ± 2

14 ± 1

28 ± 2

138 ± 10

337 ± 10

N, N, N´,N´,N´´-

0.03 µg

14 ± 4

11 ± 3

28 ± 8

117 ± 9

313 ± 25

Pentamethyl-N-

0.1 µg

17 ± 5

12 ± 4

27 ± 6

108 ± 6

326 ± 13

tallowalkyl- 1,3-

0.3 µg

15 ± 2

12 ± 5

28 ± 6

111 ± 4

317 ± 8

propanediammonium

1 µg

16 ± 5

11 ± 3

32 ± 3

112 ± 3

329 ± 21

chloride

3 µg

14 ± 1

11 ± 3

24 ± 4

95 ± 23

310 ± 18

10 µg

5 ± 3R

4 ± 1R

14 ± 3R

63 ± 18R

66 ± 4R M

33 µg

0 ± 1R M

3 ± 1M R

9 ± 1M R

0 ± 0M R

62 ± 13M R

100 µg

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

12 ± 2M R

NaN3

10 µg

1706 ± 27

1760 ± 170

4-NOPD

10 µg

464 ± 20

4-NOPD

50 µg

81 ± 6

MMS

3.0 µL

2302 ± 36

With Activation

DMSO

19 ± 4

23 ± 6

42 ± 7

129 ± 8

376 ± 19

Untreated

20 ± 3

20 ± 5

53 ± 8

139 ± 17

403 ± 41

N, N, N´,N´,N´´-

0.3 µg

19 ± 3

16 ± 1

38 ± 11

111 ± 2

355 ± 42

Pentamethyl-N-

1 µg

21 ± 6

20 ± 3

43 ± 5

114 ± 7

453 ± 53

tallowalkyl- 1,3-

3 µg

17 ± 3

21 ± 6

38 ± 3

130 ± 2

429 ± 49

propanediammonium

10 µg

20 ± 4

21 ± 1

37 ± 4

132 ± 12

447 ± 55

chloride

33 µg

18 ± 3

24 ± 2

44 ± 3

147 ± 10

397 ± 32

100 µg

18 ± 3P R

12 ± 4P R

42 ± 3P R

48 ± 4P R

284 ± 22P R

250 µg

4 ± 2P M R

9 ± 2P M R

12 ± 3P M R

16 ± 5P M R

29 ± 9P M R

500 µg

0 ± 0P M R

0 ± 0P M R

0 ± 1P M R

0 ± 0P M R

14 ± 4P M R

2-AA

2.5 µg

372 ± 12

415 ± 4

1859 ± 10

2546 ± 58

2-AA

10.0 µg

2027 ± 265

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

P

Reduced background growth

Manual count

Precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity 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.
Therefore, N, N, N´,N´,N´´-Pentamethyl-N-tallowalkyl- 1,3- propanediammonium chloride is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The test item N, N, N´,N´,N´´-Pentamethyl-N-tallowalkyl- 1,3- propanediammonium chloride was assessed for its potential to induce gene mutations ac­cording to the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. Due to strong toxic effects in the pre-experiment, the plate incorporation was repeated (reported as experiment I). Due to contamination of bacteria suspension of strains TA 1537 and TA 98 in experiment I, this part was repeated under identical conditions (reported as part of experiment I). The test item was tested at the following concentrations:

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

Experiment I and II without S9 mix:                        0.03; 0.1; 0.3; 1; 3; 10; 33; and 100 µg/plate
Experiment I and II with S9 mix:                        0.3; 1; 3; 10; 33; 100; 250; and 500 µg/plate

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 - 500

10 - 100

100 - 500

TA 1537

10 - 5000

100 - 5000

33 - 100

250 - 500

10 - 100

100 - 500

TA 98

10 - 5000

100 - 5000

33 - 100

250 - 500

10 - 100

100 - 500

TA 100

10 - 5000

100 - 5000

10 - 100

/

10 - 100

100 - 500

TA 102

10 - 5000

100 - 5000

10 - 100

/

10 - 100

100 - 500

/ = no reduced background growth

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

333 - 5000

33 - 100

250 - 500

10 - 100

250 - 500

TA 1537

33 - 5000

333 - 5000

33 - 100

250 - 500

10 - 100

250 - 500

TA 98

33 - 5000

333 - 5000

100

250 - 500

33 - 100

250 - 500

TA 100

33 - 5000

333 - 5000

33 - 100

250 - 500

33 - 100

100 - 500

TA 102

33 - 5000

333 - 5000

33 - 100

250 - 500

10 - 100

250 - 500

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with N, N, N´,N´,N´´-Pentamethyl-N-tallowalkyl- 1,3- propanediammonium chloride at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).