N-[3-(dimethylamino)propyl]docosanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:

- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (study equivalent to OECD TG 471 and GLP); S. typhimurium strains: tested up to cytotoxicity; E. coli WP 2 uvrA: tested up to limit dose

- Mammalian cell gene mutation assay with Mouse Lymphoma (L5178Y) cells (met. act.: with and without) (OECD Guideline 476 and GLP); tested up to cytotoxic concentrations; read-across: Stearic acid 3-(dimethylaminopropyl)amide

- In vitro mammalian chromosome aberration test with peripheral human lymphocyte cultures (met. act.: with and without) (OECD Guideline 473 and GLP); tested up to precipitating concentrations; read-across: Stearic acid 3-(dimethylaminopropyl)amide

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:

2005-04-05 to 2005-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(Notification No. 77 of JMOL on September 1, 1988 and Notification No. 67 of JMOL on June 2, 1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: additional rfa mutation and uvrB mutations in S. typhimurium strains and uvrA mutation in E.coli strain

Metabolic activation:

with and without

Metabolic activation system:

S9Mix

Test concentrations with justification for top dose:

Initial Assay/range-finding: 5, 20, 78, 313, 1250 and 5000 µg/plate
Confirmatory Assay: 39, 78, 156, 313, 625 and 1250 µg/plate for S. typhimurium and 156, 313, 625, 1250, 2500 and 5000 µg/plate for E. coli

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran 100% (Wako Pure Chemical Industries, Ltd., Lot number: CEH5308)
- Justification for choice of solvent/vehicle: tetrahydrofuran was used as the solvent of the test substance by the reason that it could obtain solution, and that the test substance is judged stable. It was confirmed previously that the solvent of tetrahydrofuran did not cause bad influence to growh of the bacterial strains and metabolism of drug-metabolizing enzymes

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: (without metabolic activation)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours; simultaneous with exposure

SELECTION AGENT (mutation assays): L-histidine (Salmonella tester strains), L-tryptophan (E. coli tester strain)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: S. typhimurium 1.6-3.0*10E08 cells, E.coli 6.7-7.0*10E08 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effect of the test substance was examined with a stereoscopic microscope

Evaluation criteria:

When the negative control and the positive control values satisfy acceptance criteria and
the test results satisfy acceptance criteria, the test substance was judged positive for mutagenic
activity when clear dose-related increase in the number of the revertant colonies and two-fold
or more increase in the number of the revertant colonies compared with the negative control
were observed with reappearance.

Statistics:

No statistical analysis was used for evaluation of the test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: N/A
- Effects of osmolality: N/A
- Evaporation from medium: N/A
- Water solubility: N/A
- Precipitation: precipitation of the test substance was detected at the dose of 313 µg/plate or more in both the presence and the absence of metabolic activation, but it did not have a bad influence on the observation of the revertant colonies.
- Other confounding effects: the growth of the contaminant was not observed in a result of the sterility test

RANGE-FINDING/SCREENING STUDIES:The preliminary reverse mutation test was performed to determine the most favorable dose levels of the test substance in the reverse mutation test at each of the bacterial strain. The maximum dose of the preliminary reverse mutation test was set at 5000 µg/plate in accordance with the test guideline applied, and total six different dose levels with factor of 4 from the maximum dose were employed.

COMPARISON WITH HISTORICAL CONTROL DATA: The numbers of the revertant colonies of the negative control and the positive control were within the range of the standard value of the historical data in the laboratory

ADDITIONAL INFORMATION ON CYTOTOXICITY:The test substance showed toxic effect in Salmonella typhimurium TA1535 and TA1537
of the absence of metabolic activation at the dose of 625 µg/plate or more. In the test of Salmonella typhimurium TA98 and TA100 in the absence of metabolic activation, similarly, toxic effect of the test substance was observed at the dose of 1250 µg/plate or more. Although, neither dose-related increase in the number of the revertant colonies nor two-fold or more increase in the number of the revertant colonies compared with the negative control was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Details on Cytotoxicity:

Toxic effect in S. typhimurium TA1535 and TA1537 in the absence of metabolic activation at 625 µg/plate or more. In the test of S. typhimurium TA98 and TA100 in the absence of metabolic activation toxic effect at a dose of 1250 µg/plate or more.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The mutagenic activity of DIMAPDO was examined in the reverse mutation test using S. typhimurium tester strains TA98, TA100, TA1535, and TA1537 and E. coli tester strain WP2 uvrA. The test was performed in pre-incubation methods in all bacterial strains in both the presence and the absence of metabolic activation.From the foregoing results, it is concluded that the mutagenic activity of DIMAPDO is considered negative under the test conditions employed.

Executive summary:

In a reverse gene mutation assay in bacteria in a study equivalent to OECD471, strains TA100, TA1535, TA98 and TA1537 of S. Typhimurium were exposed to DIMAPDO in tetrahydrofuran at concentrations of 39, 78, 156, 313, 625 and 1250 µg/plate and Escherichia coli WP2 uvrA were exposed to 156, 313, 625, 1250, 2500 and 5000 µg/plate.

The test was performed with the pre-incubation method in all bacterial strains in both the presence and the absence of metabolic activation.

 

DIMAPDO was tested up to cytotoxic concentrations for S. typhimurium (1250 µg/plate) and to limit concentration (5000 µg/plate) for E.coli. Precipitation of the test substance was detected at a dose of 313 µg/plate or more in both the presence and the absence of metabolic activation, but it did not have a bad influence on the observation of the revertant colonies. The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: tertiary amines, amides, and fatty acid chains with comparable length and degree of saturation (corresponding to scenario 2 of the read-across assessment framework)

The read-across hypothesis is based on structural similarity of target and source substances. The target and source chemicals have a very similar structure in that they are comprised of a hydrophobic (alkyl) and hydrophilic (amine headgroup) end. Due to this motif they form micelles (colloidal dispersions) and have surfactant properties.
Based on available experimental data, including key physicochemical properties and data from genotoxicity studies, the read-across strategy is supported by a similar toxicological profile of all substances.

Therefore, read-across from the existing toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

A justification for read-across is attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to IUCLID section 13.

4. DATA MATRIX
See justification for read-across attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix > 4 µg/mL, with S9 > 50 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Based on the results obtained with the source substance, DIMAPDO is not mutagenic in the in vitro gene mutation study in mammalian cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: tertiary amines, amides, and fatty acid chains with comparable length and degree of saturation (corresponding to scenario 2 of the read-across assessment framework)

The read-across hypothesis is based on structural similarity of target and source substances. The target and source chemicals have a very similar structure in that they are comprised of a hydrophobic (alkyl) and hydrophilic (amine headgroup) end. Due to this motif they form micelles (colloidal dispersions) and have surfactant properties.
Based on available experimental data, including key physicochemical properties and data from genotoxicity studies, the read-across strategy is supported by a similar toxicological profile of all substances.

Therefore, read-across from the existing toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

A justification for read-across is attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to IUCLID section 13.

4. DATA MATRIX
See justification for read-across attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: peripheral human lymphocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

Based on the results obtained woth the source substance, DIMAPDO is not clastogenic in the in vitro mammalian chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Not all data on genetic toxicity are available data for the target substance DIMAPDO. For the assessment of effects of DIMAPDO on mutagenicity results from the following studies are taken into consideration:

- an equivalent to OECD 471 reverse gene mutation assay in bacteria of the target substance DIMAPDO

- an OECD 473 in vitro mammalian chromosome aberrationand an OECD 476 mammalian gene mutation assay of the source substance Stearic acid 3-(dimethylaminopropyl)amide

- an additional equivalent to OECD 471 reverse gene mutation assay of thesource substance Stearic acid 3-(dimethylaminopropyl)amide for the justification for read across for the endpoint genetic toxicity

A justification for read-across is attached to IUCLDI section 13. 

 

Reverse gene mutation assays in bacteria

In a reverse gene mutation assay in bacteria in a study equivalent to OECD TG 471, strains TA100, TA1535, TA98 and TA1537 of S. typhimurium were exposed to the target substance DIMAPDO in tetrahydrofuran at concentrations of 39, 78, 156, 313, 625 and 1250 µg/plate and Escherichia coli WP2 uvrA were exposed to156, 313, 625, 1250, 2500 and 5000 µg/plate.

The test was performed with the pre-incubation method in all bacterial strains in both the presence and the absence of metabolic activation.

 

DIMAPDO was tested up to cytotoxic concentrations for S. typhimurium (1250 µg/plate) and to limit concentration (5000 µg/plate) for E.coli. Precipitation of the test substance was detected at a dose of 313 µg/plate or more in both the presence and the absence of metabolic activation, but it did not have a bad influence on the observation of the revertant colonies. The positive controls induced the appropriate responses in the corresponding strains.There wasno evidenceof induced mutant colonies over background.

 

For the structurally similar source substance Stearic acid 3-(dimethylaminopropyl)amide (a.i. 85 %) a reverse gene mutation assay in bacteria equivalent to OECD guideline 471, with strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) was negative. For S. typhimurium concentrations of 5, 10, 25, 50, and 75 µg/plate in the absence of mammalian metabolic activation, and at concentrations of 25, 50, 75, 100 and 250 µg/plate in the presence of mammalian metabolic activation were tested. Strain E. coli WP2 uvrA was tested at concentrations of 10, 25, 50, 75, and 100 µg/plate in the absence and at 50, 75, 100, 250, and 500 µg/plate in the presence of metabolic mammalian activation.

Two tests were performed, the first test using the plate incorporation and the second test the preincubation method. In the first mutation assay (plate incorporation) Stearic acid 3-(dimethylaminopropyl)amide did not induce a significant dose-related increase in the number of revertant colonies in all five tested strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in the second mutation assay (preincubation method). No precipitation was observed.

Cytotoxic effects of the test substance were observed in the preincubation assay in allS. typhimurium strains, at a concentration of ≥ 50 µg/plate in the absence of mammalian metabolic activation. Additionally in a range finding test cytotoxic effects were shown at ≥ 500 µg/plate in the E. coli. WP2 uvr A strain.

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed.Under the conditions of the study, Stearic acid 3-(dimethylaminopropyl)amide was negative for mutagenic potential.

 

 

Mammalian cell gene mutation assay

Data for the source substance Stearic acid 3-(dimethylaminopropyl)amide is available from a mammalian cell gene mutation assay (thymidine kinase locus) according to OECD Guideline 476.

L5178Y mouse lymphoma cells cultured in vitro were exposed to Stearic acid 3-(dimethylaminopropyl)amide (100% purity) in ethanol in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

First experiment

Without S9-mix, 3 h treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2.5 and 5μg/mL

With 8% S9-mix, 3 h treatment: 0.1, 0.6, 1, 5, 10, 20, 30 and 40μg/mL

Second experiment

Without S9-mix, 3 h treatment: 0.01, 0.03, 0.1, 0.3, 0.6, 1 and 3μg/mL

With 12% S9-mix, 24 h treatment: 0.1, 1, 3, 10, 30, 40, 50 and 60μg/mL

Stearic acid 3-(dimethylaminopropyl)amide was tested up to cyctotoxic concentrations.The positive controls induced the appropriate response. This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

The results of the L 5178Y/TK Mouse Lymphoma Mutagenesis Assay indicate that, under the conditions of this study Stearic acid 3-(dimethylaminopropyl)amide did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative.

 

Mammalian cell cytogenetics assay

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473, adopted 21 July 1997 and EU Method B.10, May 2008, peripheral human lymphocyte cultures were exposed to the source substance Stearic acid 3-(dimethylaminopropyl)amide in ethanol at the following concentrations:

First experiment:

without and with S9-mix (3 h exposure time, 24 h fixation time): 0, 1, 3 and 10 μg/mL

Second experiment:

without S9-mix (24 h and 48 h exposure time, 24 h and 48 h fixation time): 0, 3, 6, 10, 15, 20 and 25 μg/mL

with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10 μg/mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Stearic acid 3-(dimethylaminopropyl)amide was tested up to precipitating concentrations (10 µg/mL).

Both in the absence and presence of S9-mix Stearic acid 3-(dimethylaminopropyl)amide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Stearic acid 3-(dimethylaminopropyl)amide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Stearic acid 3-(dimethylaminopropyl)amide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.There was no evidence of chromosome aberrations induced over background.

This study is classified as acceptable andsatisfies the requirement for OECD Test Guideline 473 forin vitrocytogenetic mutagenicity data. 

 

Based on the available reliable, relevant and adequate data, there is no evidence of genotoxicity for DIMAPDO in vitro. There are no data gaps for the endpoint genotoxicity. In conclusion, there is no need to carry out in vivo tests for genetic toxicity. 

Justification for classification or non-classification

There was no evidence for any genotoxic intrinsic properties in the Ames-Test conducted with the substance itself and in a mouse lymphoma assay and a chromosome aberration study conducted with a structurally closely related substance. DIMAPDO is therefore considered to be non-genotoxic. DIMAPDO is not to be classified as genotoxic according GHS Regulation EC No 1272/2008.o labelling is required.