Amides, C12-18, N-[3-(dimethylamino)propyl]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cocoamidopropyldimethylamine was tested in three in vitro genotoxicity studies (bacterial reverse mutation test, mammalian chromosomal aberration test, mammalian cell gene mutation) to current protocol and carried out to GLP with well defined test substance.  All three tests were negative.

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:

September 07, 2009 - December 24, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant and according to guidelines stated.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Please see below

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Please see below

Principles of method if other than guideline:

- on one occasion, the test item was stored without nitrogen gas for about 2 hours. Based on information supplied by the Sponsor, this deviation was not considered to have compromised the validity or integrity of the study,
- the cultures were put into 13 mL of nutrient broth (as described in CIT’s SOP) instead of 6 mL. This minor deviation was not considered to have compromised the validity or integrity of the study,
- the overlay agar was maintained at 50°C instead of 45°C. This minor deviation was not considered to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

No details supplied

Species / strain / cell type:

other: Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37°C for about 14 hours, to produce bacterial suspensions.

Additional strain / cell type characteristics:

other: Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. Please see materials and methods for more information

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

The test item was dissolved in the vehicle at concentrations of:
100 mg/mL for the preliminary toxicity test,
10 mg/mL for the first experiment,
10, 5 and 1 mg/mL for the second experiment,
5 mg/mL for the third experiment.

Vehicle / solvent:

The vehicle was dimethylsulfoxide (DMSO), batch Nos. K39250750841 and K39250750906 (VWR, Fontenay Sous Bois, France).

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control was tested both with and without S9 mix

True negative controls:

not specified

Positive controls:

yes

Remarks:

Dissolved in dimethylsulfoxide to check the sensitivity of the test system

Positive control substance:

sodium azide

Remarks:

without S9 mix. Used on the TA 1535 and TA 100 strains. Migrated to IUCLID6: 1 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control was tested both with and without S9 mix

True negative controls:

not specified

Positive controls:

yes

Remarks:

Dissolved in dimethylsulfoxide to check the sensitivity of the test system

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix. Used on the TA 1537 strain. Migrated to IUCLID6: 50 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control was tested both with and without S9 mix

True negative controls:

not specified

Positive controls:

yes

Remarks:

Dissolved in dimethylsulfoxide to check the sensitivity of the test system

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix. Used on the TA 98 strain. Migrated to IUCLID6: 0.5 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control was tested both with and without S9 mix

True negative controls:

not specified

Positive controls:

yes

Remarks:

Dissolved in distilled water to check the sensitivity of the test system

Positive control substance:

mitomycin C

Remarks:

without S9 mix. Used on the TA 102 strain. Migrated to IUCLID6: 0.5 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control was tested both with and without S9 mix

True negative controls:

not specified

Positive controls:

yes

Remarks:

Dissolved in dimethylsulfoxide to check the sensitivity of the test system

Positive control substance:

other: 2-Anthramine

Remarks:

with S9 mix. 2 µg/plate was used for TA 1535, TA 1537 and TA 98 strains. 10 µg/plate was used for TA 102 strain.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control was tested both with and without S9 mix

True negative controls:

not specified

Positive controls:

yes

Remarks:

Dissolved in dimethylsulfoxide to check the sensitivity of the test system

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix. Used for the TA 102 strain Migrated to IUCLID6: 5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preliminary test, all experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

The direct plate incorporation method was performed as follows: test item solution (0.05 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

The preincubation method was performed as follows: test item solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Manual counting was used as needed.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 to 72 hours of incubation at 37°C

NUMBER OF REPLICATIONS:
In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
at least five dose-levels of the test item, the vehicle control, the appropriate positive control.

In a third experiment, using three plates/dose-level, the TA 1537 strain was tested without S9 mix, with:
at least five dose-levels of the test item,
the vehicle control,
the appropriate positive control.

The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

DETERMINATION OF CYTOTOXICITY
- Method: other: Number of revertants per plate were scored.

Evaluation criteria:

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive controls is higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and is consistent with the historical data of the testing facility.

Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

Not relevant

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The sterility of the test item (stock preparation) was checked and found satisfactory.

Preliminary test:
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL. Consequently, with a treatment volume of 50 µL/plate, the selected dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

A moderate precipitate was observed in the Petri plates when scoring the revertants at 5000 µg/plate without S9 mix and a moderate to strong precipitate was noted at dose-levels ≥ 1000 µg/plate with S9 mix.

A strong toxicity was noted in the three strains at dose-levels ≥ 100 µg/plate without S9 mix and ≥ 500 µg/plate with S9 mix.

Mutagenicity Experiments:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
The selected treatment-levels were:
3.13, 6.25, 12.5, 25, 50 and 100 µg/plate for all the strains in the first experiment,
1.56, 3.13, 6.25, 12.5, 25 and 50 µg/plate for all the strains in the second experiment,
6.25, 12.5, 18.8, 25, 37.5 and 50 µg/plate for the TA 1537 strain in the third experiment.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity was noted at dose-levels ≥ 25 µg/plate in the TA 100 and TA 102 strains and ≥ 50 µg/plate in the TA 1535, TA 1537 and TA 98 all strains.

A slight increase in the number of revertants, exceeding the threshold of 3-fold the corresponding vehicle control value, was noted in the second experiment in the TA 1537 strain. This increase was not considered to be biologically relevant as it was not observed in the first experiment and was not reproduced in the third experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the other four strains.

Experiments with S9 mix
The selected treatment-levels were:
15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for all the strains in the first experiment,
7.81, 15.6, 31.3, 62.5, 125 and 250 µg/plate for all the strains in the second experiment.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity was noted at dose-levels ≥ 62.5 μg/plate in the TA 102 strain, ≥ 125 μg/plate in the TA 1537, TA 98 and TA 100 strains and ≥ 250 μg/plate in the TA 1535 strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, the test item Cocoamidopropyldimethylamine did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. Based on these results, the test item does not require classification according to Directive 67/548/EEC or Regulation EC No. 1272/2008.

Executive summary:

The objective of this study was to evaluate the potential of the test item Cocoamidopropyldimethylamine to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

Methods

A preliminary toxicity test was performed to define the dose-levels of Cocoamidopropyldimethylamine to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item Cocoamidopropyldimethylamine was dissolved in dimethylsulfoxide (DMSO).

The dose-levels of the positive controls were as follows:

without S9 mix

- 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

- 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

- 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

- 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

     

with S9 mix

- 2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537 and TA 98 strains,

- 5 µg/plate of Benzo(a)pyrene (BAP): TA 100 strain,

- 10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

      

Results

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix

The selected treatment-levels were:

- 3.13, 6.25, 12.5, 25, 50 and 100 µg/plate for all the strains in the first experiment,

- 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/plate for all the strains in the second experiment.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity was noted at dose-levels  ≥ 50 µg/plate instrains.

The test item did not induce any increase in the number of revertants, which could be considered as biologically relevant, in any of the five strains.

Experiments with S9 mix

The selected treatment-levels were:

- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for all the strains in the first experiment,

- 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/plate for all the strains in the second experiment.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity was noted at dose-levels ≥ 125 µg/plate in the TA 102 strain, ≥ 125 µg/plate in the TA 1537, TA 98 and TA 100 strains and ≥ 250 µg/plate in the TA 1535 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

Conclusion

Under our experimental conditions, the test item Cocoamidopropyldimethylamine did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Under the conditions of this study, the test substance was not considered to be mutagenic and as such, does not require classification according to Regulation EC No. 1272/2008 and Directive 67/548/EEC.

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:

2009-09-04 to 2010-01-14

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

See below

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

- on one occasion, the test item was stored without nitrogen gas for about 2 hours. Based on information supplied by the Sponsor, this deviation was not considered to have compromised the validity or integrity of the study,
- to prevent any invalidation of an assay because of a possible technical problem, contamination, haemolysis or variation of the response of the positive controls, duplicate cultures of the positive controls have been performed in each experiment. The fixed cells were spread on slides and cells in mitosis were scored, but the metaphase analysis were not performed since the conditons described above were not met. except in the second experiment without S9 mix in which one out of the two cultures was replaced due to a doubtful quality of the metaphases. These minor deviations were not considered to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Origin: primary cell cultures prepared from whole blood samples from two healthy donors
- Type and identity of media: RPMI 1640 medium containing 20% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and phytohemagglutinin (mitogen)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver post-mitochondrial fraction and cofactors of rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

- First experiment (with and without S9 mix)
Mitotic index: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL,
Chromosome aberration (metaphase analysis): 3.13, 6.25 and 12.5 µg/mL

- Second experiment (with and without S9 mix)
Mitotic index: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 µg/mL
Chromosome aberration (metaphase analysis): 3.13, 6.25 and 12.5 µg/mL for 20h treatments and 12.5 µg/mL for 44h treatments.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix used at a final concentration of 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continous treatment)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix used at final concentrations of 12.5 µg/mL and 25 µg/mL.

Details on test system and experimental conditions:

- Preincubation period: 48h (at 37°C)
- Exposure duration:
First experiment: 3h
Second experiment: without S9 mix continuously until harvesting; with S9mix 3h
- Fixation time (start of exposure up to harvest of cells):
First experiment: 20h (approximately 1.5 normal cell cycles)
Second experiment: 20h and 44h (respectively 1.5 normal cell cycles and 24 hours later)

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution (10 µg/mL), treatment one and a half hours before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED:
Cytotoxicity: 1000 cells evaluated
Chromosome aberration: 200 metaphases in total (i.e. 100 metaphases/culture). When at least 10% cells with structural chromosome aberration were observed only 50 metaphases/culture were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER:
- Cell culture preparation: 0.5 mL of heparinized whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, 2mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and phytohemagglutinin (mitogen)
- Preparation of microscope slides: after treatment with spindle inhibitor each culture recieved a hypotonic treatment (KCl 0.075 M). Afterwards cells were fixed in a methanol/acetic acid mixture (3/1; v/v) and spread on glass slides before staining
- All metaphase analyses were performed blind
- Structural aberrations recorded: gaps, chromatid and chromosome breaks and exchanges, multiple aberrations and pulverizations

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

The frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, these results were compared using a chi-square test in which p = 0.05 was used as the lowest level of significance

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: no, osmolality was 465 mOsm/kg H2O in vehicle control, while in dose-level of 5000 µg/mL it was 400 mOsm/kg H2O. .
- Solubility in
vehicle: freely soluble at 55 mg/mL
- Precipitation: no precipitate in the culture medium at the highest dose-level of 5000 µg/mL
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was within the range provided by historical data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

- Frequency of cells with structural chromosome aberrations for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

-Metaphase analysis

In the first experiment (3 -hour treatment), the dose-level of 12.5 µg/mL was selected for the metaphase analysis mistake. Indeed, this dose-level of 12.5 µg/mL was selected for the metaphase analysis by mistake. Indeed, this dose-level showed an important decrease in the mitotic index indicating a high level of cytotoxicity which may lead to false positive results. nevertheless, in the present study, no significant increase in the frequency of cells with sturctural chromosomal aberrations was observed at this dose-level (despite the high level of toxicity) or at the two other analysed dose-levels.

For both experiments with and without S9 mix no significant increase in the frequency of cells with structural chromosomal aberrations (such as chromatid-type or chromosome-type deletions or exchanges) was noted for either of the 20 or the 44 hour treatments and for either of the harvest times.

Conclusions:

Interpretation of results (migrated information):
negative

Under these experimental conditions, no statistically significant increase in the frequency of cells with chromosome aberrations was observed, both with and without S9 mix, using a dose range from 0.39 to 50 µg/mL. Cocoamidopropyldimethylamine did not show any clastogenic activity in the in vitro mammalian chromosome aberration test with cultured human lympocytes.

Executive summary:

The potential of cocoamidopropyldimethylamine to induce chromosome aberrations in cultured human lymphocytes was assessed according to the OECD guideline 473 and the EU Method B.10. The study was conducted in compliance with the principles of Good Laboratory Practice.

Two experiments, with and without a metabolic activation system, were performed in duplicate and together with vehicle and positive controls. In the first experiment cultured human lympocytes were exposed to 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL of cocoamidopropyldimethylamine for 3h and harvested 20 hours after the beginning of treatment. Cytotoxicity was recorded for all these concentrations, while only concentrations 3.13, 6.25 and 12.5 µg/mL were selected for the metaphase analysis. Since this first experiment gave negative results both with and without activation, a second experiment was performed as stated in the guidelines. In this second experiment, treatment in absence of S9 mix was continuous until harvest whilst cultured cells in the presence of S9 mix were exposed for 3 hours to sodium hypophosphite. Cells were always harvested 20 and 44 hours after the beginning of the treatment. The concentrations of test substance used in the second experiment were 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 µg/mL to calculate the mitotic index and for metaphase analysis cocoamidopropyldimethylamine concentrations of 3.13, 6.25 and 12.5 µg/mL was selected for the 20h treatments and 12.5 µg/mL for the 44h treatments.

The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

cocoamidopropyldimethylamine did not exhibit significant cytotoxicity at the dose-levels assessed (except at the dose-level of 12.5 µg/mL for 3 -hour treatment indicating a high level of cytotoxicity) and did not induce any statistically significant increases in the frequency of cells with aberrations in either of the two experiments for all tested concentrations (range: 0.39 -50 µg/ml).

Under these test conditions cocoamidopropyldimethylamine did not show any clastogenic activity in thein vitromammalian chromosome aberration test with cultured human lympocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2011 - 25 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix
Using a treatment volume of 1%, the selected dose-levels were as follows:
.           0.63, 1.25, 2.5, 5, 7.5 and 10 µg/mL for the first experiment (3-hour treatment),
.           0.16, 0.31, 0.63, 1.25, 2.5 and 5 µg/mL for the second experiment (24‑hour treatment).

Experiments with S9 mix
Using a treatment volume of 1%, the selected dose-levels were 2.5, 5, 10, 20, 30 and 40 µg/mL for both experiments (3-hour treatment).

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: The test item was found freely soluble in the dimethylsulfoxide (DMSO).
Using a stock solution at 500 mg/mL and a treatment volume of 1% in culture medium, it was possible to reach the dose-level of 5000 µg/mL which is the highest dose-level recommended in the international guidelines. DMSO was therefore selected as the vehicle to be used in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:

Positive result defined as:
- At least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 E-6
- A dose-related trend is demonstrated by a statistically significant trend test
- Unless clearly positive, the reproducibility should be confirmed

Negative results defined as:
- No evidence of mutagenicity at concentrations inducing moderate cytotoxicity (10% < Adj. RTG <20%), or
- If no culture has 10% < Adj. RTG <20%:
* at least one negative data point with 20% < Adj. RTG <25% + negative data from 20% < Adj. RTG <100%, or
* at least one negative data point with 1% < Adj. RTG <10% + negative data from 25% < Adj. RTG <100%

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

maximum doses exhibited growth inhibition

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item, Coco amidopropyldimethylamine, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Coco amidopropyldimethylamine, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells.

The study was performed according to international guidelines (OECD No. 476 and Council regulation (EC) No. 440/2008 of 30 May 2008) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After two preliminary toxicity tests, the test item Coco amidopropyldimethylamine, was tested in two independent mutagenicity experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

 

Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) andCloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

The Cloning Efficiencies (CE₂), the mutation frequencies and the suspension growths of the vehicle controls were mainly as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

 

Since the test item was found toxic in the preliminary tests, the choice of the highest dose‑level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).

Experiments without S9 mix

Using a treatment volume of 1%, the selected dose-levels were as follows:

.           0.63, 1.25, 2.5, 5, 7.5 and 10 µg/mL for the first experiment (3-hour treatment),

.           0.16, 0.31, 0.63, 1.25, 2.5 and 5 µg/mL for the second experiment (24‑hour treatment).

 

Cytotoxicity

Following the 3-hour treatment, a moderate to marked toxicity was induced at dose-levels ≥ 7.5 µg/mL, as shown by a 49 to 79% decrease in the Adj. RTG.

Following the 24-hour treatment, a marked to severe toxicity was induced at dose‑levels ≥ 2.5 µg/mL, as shown by a 66 to 100% decrease in the Adj. RTG.

Mutagenicity

Following the 3-hour and 24-hour treatments, no noteworthy increase in the mutation frequency was observed in comparison to the vehicle control at any of the tested dose-levels. Moreover, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 10 µg/mL and 2.5 µg/mL for the first and second experiments, respectively did not demonstrate a dose-response relationship (p > 0.05).These results met the criteria for a negative response.

 

Experiments with S9 mix

Using a treatment volume of 1%, the selected dose-levels were 2.5, 5, 10, 20, 30 and 40 µg/mL for both experiments (3-hour treatment).

 

Cytotoxicity

In the first and second experiments, a severe toxicity was induced at dose-levels ≥ 30 µg/mL, as shown by a 90 to 100% decrease in the Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control at any of the tested dose-levels in either experiment. Moreover, a statistical analysis was performed on the individual mutation frequencies of total induced mutants obtained in the first and second experiments at dose-levels up to 30 and 20 µg/mL, respectively. This analysis did not demonstrate a linear trend between the mutation frequency and the dose (p > 0.05).These results met the criteria for a negative response.

 

Conclusion

The test item, Cocoamidopropyldimethylamine, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genotoxicity by Cocoamidopropyldimethylamine is not expected. In physiological circumstances, Cocoamidopropyldimethylamine has a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

Cocoamidopropyldimethylamine was tested for genetic toxicity in three in vitro tests. In a bacterial reverse mutation assay the substance was not mutagenic with or without S9 mix up to 50 µg/mL (the highest dose-level achievable regarding the severe cytotoxicity observed at higher dose-levels). It was also tested for mammalian cell gene mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells and was not mutagenic in concentrations up to 30 µg/mL (based on the level of toxicity). In addition, the substance was tested in a chromosomal aberration test in human lymphocytes and was found not to be clastogenic up to 12.5 µg/mL (despite the high level of toxicity observed at this dose-level). All tests were to current OECD/EU protocols carried out to GLP with a clearly defined and described test substance. Based on these results it can be concluded that cocoamidopropyldimethylamine is not expected to be a genotoxic hazard to human health.

Justification for classification or non-classification

Available studies show no concern for possible genotoxicity. Also further property data for Cocoamidopropyldimethylamine indicate that genotoxic properties are rather unlikely.