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Diss Factsheets

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:
September 07, 2009 - December 24, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant and according to guidelines stated.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[3-(dimethylamino)propyl] C 12-C18 alkylamide
EC Number:
932-121-8
Cas Number:
1147459-12-8
Molecular formula:
UVCB substance not applicable
IUPAC Name:
N-[3-(dimethylamino)propyl] C 12-C18 alkylamide
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Coco amidopropyldimethylamine
- Substance type: Amides, C12-18 (even numbered), N-[3-(dimethylamino)propyl]
- Physical state: Light brown, paste to solid
- Analytical purity: 99.1%
Free dimethyl amino propylamine 0.4%
Free fatty acid 0.5%
- Purity test date: 31 August 2009
- Lot/batch No.: S001824
- Expiration date of the lot/batch: 2nd July 2019
- Storage condition of test material: At room temperature, under nitrogen gas, in a dry and well-ventilated room
- Other: Kept in two transparent glass flasks.

Method

Target gene:
No details supplied
Species / strain
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).
Controlsopen allclose all
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

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.