Amides, C16-18, N-[3-(dimethylamino)propyl]

Endpoint summary

Additional information

A reverse gene mutation assay in bacteria according to OECD guideline 471 is available for the target substance C16-18 DMAPA amidoamine.A mammalian cell gene mutation assay, and an in vitro mammalian chromosome aberration test are available for the structurally closely related source substance Stearic acid 3-(dimethylaminopropyl)amide. A justification for read-across is given below.

 

The target substanceC16-18 DMAPA amidoamine is not mutagenic in the Salmonella typhimurium reverse mutation assay. The source substance Stearic acid 3-(dimethylaminopropyl)amide is not mutagenic in the Salmonella typhimurium reverse mutation assay and the mammalian cell gene mutation assay (TK test) using mouse lymphoma L5178Y cells. In an in vitro mammalian cell cytogenetics assay Stearic acid 3-(dimethylaminopropyl)amide did not induce structural chromosomal aberrations in cultured peripheral human lymphocytes. Based on the available data, there was no evidence of genotoxicity for the target substance C16-18 DMAPA amidoamine.

Also DMAPA did not show any alerts for genotoxic potential: DMAPA was not mutagenic in the Ames test and didnot induce micronuclei in normochromatic erythrocytes from mouse bone marrow cells(NTP; OECD SIDS). All other study reports on genotoxicity published in the ECHA registered substance database did not raise any concern for genotoxic properties of DMAPA.

In conclusion, there is no need to carry out in vivo tests for genetic toxicity. There are no data gaps for this endpoint. No human data are available for genetic toxicity. However, there is no reason to believe that the negative results would not be relevant to humans.

 

Bacterial reverse gene mutation assays

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to C16-18 DMAPA amidoamine (Amides, C16-18 (even numbered), N-[(dimethylamino)propyl]) (100% a.i.) in THF at concentrations of 0 (control), 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate in the first experiment with plate incorporation; 0 (control), 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate in the second experiment with salmonella strains, and 3; 10; 33; 100; 333; 1000; 2500; and 5000μg/plate with E. coli using the pre-incubation method in the presence and absence of mammalian metabolic activation (rat liver S9 mix). Experiment IIa was performed without S9 mix in salmonella strains TA 1535, TA 98, TA 100: 0.1; 0.3; 1; 3; 10; 33; 100; 333μg/plate.

Amides, C16-18 (even numbered), N-[(dimethylamino)propyl] was tested up to cytotoxic concentrations. Cytotoxic effects were noted starting at 10 µg/plate in strains TA 1535, TA 98 and TA 100 and in strain 1537 at 33 µg/plate without metabolic activation. With metabolic activation, cytotoxic effects were seen starting at 333 µg/plate in strain TA 98 and starting at 1000 µg/plate in strains TA 1535, TA 1537 and TA 98. In E. coli WP 2 uvrA cytotoxic effects were seen at 1000 µg/plate without S9 mix and at 2500 µg/plate with metabolic activation.

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I and II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 μg/plate in experiment I and from 1000 to 5000 μg/plate in experiment II. The undissolved particles had no influence on the data recording.

The positive controls induced the appropriate responses in the corresponding strains.The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.There was no evidence of induced mutant colonies over background.

 

In a reverse gene mutation assay in bacteria equivalent to OECD guideline 471, strains of S. Typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to Stearic acid 3-(dimethylaminopropyl)amide (a.i. 85 %) for S. typhimurium at 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. Strain E. coli WP2uvrA 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 all S. Typhimurium strains, at a concentration of >/= 50 µg/plate in the absence of mammalian metabolic activation. Additionally at 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.There was no evidence of induced mutant colonies over background.

 

Mammalian cell gene mutation assay

In a mammalian cell gene mutation assay according to OECD guideline 476, adopted July 21, 1997 (thymidine kinase (TK)),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 5 μg/mL

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

Second experiment

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

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

Stearic acid 3-(dimethylaminopropyl)amide was tested up to cyctotoxic concentrations. The positive controls induced the appropriate response.There was no evidence of induced mutant colonies over background.

 

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 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 10μg/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 25μg/mL; with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10μg/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.

 

 

Justification for read-across

1. Read-across hypothesis and justification

This read-across is based on the hypothesis that source and target substances have similar toxicological properties because

·        they are manufactured from similar resp. identical precursors under similar conditions

·        the metabolism pathway leads to comparable products (amine backbone and long chain fatty acids) and non-common products predicted to have no toxicological effects (long chain fatty acids).

 

Therefore, read-across from the existing genotoxicity studies on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VII 8.4.1, Annex VIII 8.4.2, and 8.4.3 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

The justification of the proposed read-across approach is elaborated in the next chapters.

 

 

2. Justification for read-across

 

2.1 Substance Identity

 

Table 1: Substance identities

	Source substance	Target substance
	Stearic acid 3-(dimethylaminopropyl)amide	C16-18 DMAPA amidoamine
	mono constituent substance	UVCB substance
CAS number	7651-02-7
Chain length distribution	< C16: < 1.6% C16: < 7% C18: > 89.8% > C18: < 1.6%	C14: <= 5 % C16: 25-35 % C18: >= 61 %
DMAPA	<0.002%	<=0.01%

 

2.1 Substance Identity

Substance descriptions

The target substanceC16-18 DMAPA amidoamine is a UVCB substance manufactured from saturated C16-18 fatty acids and N, N-dimethylpropylenediamine (DMAPA). It is composed of C16 and C18 amides of DMAPA, with C18 being the larger part (>/= 61%)

 

The source substanceStearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid andN, N-dimethylpropylenediamine. It is composed of mainly C18 amides (> 89.8%) of DMAPA and small amounts of the C16 amide (<7%).

 

2.2 Common breakdown products

The source substance Stearic acid 3-(dimethylaminopropyl)amide is the main component of the UVCB target substanceC16-18 DMAPA amidoamine. The only difference is the chain length distribution: the target substance also contains a significant amount of the C16 amide.

This is not considered to be of relevance for metabolism.Both substances are amides which after resorption may be hydrolysed by amidases resulting in free fatty acids and DMAPA. The carboxylic acids then are further degraded by the mitochondrial beta-oxidation process (for details see common text books on biochemistry). The fatty acids enter normal metabolic pathways and are therefore indistinguishable from fatty acids from other sources including diet. The amine compounds are not expected to be further metabolised, but excreted via the urine mainly unchanged. 

 

2.3 Differences

The slight differences in fatty acid chain length (higher percentage of C16 in the target substance vs. corresponding higher percentage C18 in the source substance) are not considered to be of relevance for genotoxicity.

 

3. Physicochemical properties:

Table 2: Physicochemical properties

Endpoints	Source substanceStearic acid 3-(dimethylaminopropyl)amide	Target substanceC16-18 DMAPA amidoamine
Molecular weight	368.64 g/mol	340.59 - 368.64
Physical state at 20°C / 1013 hPa	Solid (paste)	Solid (waxy)
Melting point	OECD TG 102, RL1, non-GLP 67.4°C	OECD TG 102, RL1, non-GLP 41.8°C
Boiling point	OECD TG 103, RL1, non-GLP 412.3°C	OECD TG 103, RL1, non-GLP 320.5°C
Surface tension	ISO 4311, plate method, RL1, non-GLP 37.86 mN/m at 0.22 g/L	OECD TG 115, ring method, RL1, non-GLP 26.7 mN/m at 2.7 mg/L
Water solubility	OECD TG 105, RL1, non-GLP 10 mg/L at 20°C	OECD guideline 105/EU method A.6, slow stirring method/HPLC, RL1, non-GLP 3.65 mg/L at 23°C
Log Kow	---	Calculation (ACD/Labs Release 12.00, Product version 12.01 ) >6.6
	EU method A.8, calculation based on solubility in n-Octanol and water; RL2, non-GLP 2.01 at 20°C, pH7	Read-across from Stearic acid 3-(dimethylaminopropyl)amide
Vapour pressure	OECD TG 104, RL1, ISO17025 compliance 3.4E-08 Pa at 20°C	Read-across from Stearic acid 3-(dimethylaminopropyl)amide

 

 

4. Comparison of data from human health endpoints

4.1 Toxicity data of the target and source substances

Table 3: General toxicological profiles forStearic acid 3-(dimethylaminopropyl)amide andC16-18 DMAPA amidoamine

Endpoints	Source substance Stearic acid 3-(dimethylaminopropyl)amide	Target substanceC16-18 DMAPA amidoamine
Acute toxicity oral	OECD TG 423, RL1,GLP   LD50(rat) > 2000 mg/kg bw	OECD TG 423, RL1,GLP   LD50(rat) > 2000 mg/kg bw
Eye irritation	OECD TG 405, RL1, GLP   Category 1 (irreversible effects on the eye)	No data; read-across
	OECD TG 437, RL1, GLP   not severely irritating /not corrosive	No data; read-across
Skin irritation	OECD TG 439, RL1, GLP   not irritating	OECD TG 439, RL1, GLP   not irritating
	OECD TG 404, RL1, GLP   not irritating	No data; read-across
Sensitisation	OECD TG 406 (GPMT), RL1, GLP   not sensitising	No data; read-across
Genotoxicity	OECD TG 471 (Ames test), RL1, GLP   Negative	OECD TG 471, RL1, GLP   Negative
	OECD TG 467 (MLY), RL1, GLP   Negative	No data; read-across
	OECD TG 473 (Chromosome aberrations), RL1, GLP   Negative	No data; read-across
Repeated dose toxicity oral	Similar to OECD TG 407 (14 d DRF), rat, RL1, GLP   clinical signs/mortality (all animals at 500 mg/kg bw/d were sacrificed for humane reasons); haematology (slightly lower red blood cell and higher reticulocyte counts in males at 50 and 200 mg/kg bw/d); clinical biochemistry (higher ALAT activity in 2 males at 50 mg/kg bw/d, 2 males and 1 female at 200 mg/kg bw/d, higher alkaline phosphatase activity in 1 female at 200 mg/kg bw/d, higher potassium level in males at 50 and 200 mg/kg bw/d)	No data; read-across
Repeated dose toxicity dermal	Similar to OECD TG 411, rabbit, RL2, GLP   NOAEL(systemic) = 200 mg/kg bw/d (highest dose administred)	No data; read-across
Reproduction / Developmental Toxicity Screening Test	OECD TG 421, rat, RL1, GLP   NOAEL(parental)= 70 mg/kg bw/d; NOAEL(fertility females)= 70 mg/kg bw/d; NOAEL (fertility males) = 200 mg/kg bw/d; NOAEL(development)= 200 mg/kg bw/d	No data; read-across
Prenatal developmental toxicity	Similar to OECD TG 414, rabbit, RL2, GLP   NOAEL(development)=200 mg/kg bw/d (highest dose administered)	No data; read-across

 

Experimental data for the target substance C16-18 DMAPA amidoamine are available for acute oral toxicity, skin irritation (in vitro) and genotoxicity (bacterial reverse mutation assay).

No experimental data are available for the target substance C16-18 DMAPA amidoamine concerning the endpoints eye irritation, sensitisation, repeated dose toxicity, and reproductive/developmental toxicity. However, as demonstrated above, the source substance Stearic acid 3-(dimethylaminopropyl)amide is the main constituent of the target substance. The additional minor constituents with differing fatty acid chain lengths are not considered to influence the outcome of the genotoxic studies.

This is supported by the similar results observed in the available studies: Both, the target substance C16-18 DMAPA amidoamine and the source substanceStearic acid 3-(dimethylaminopropyl)amide are of low acute toxicity when administered orally. The LD50 for both substances was > 2000 mg/kg bw. In both studies, 2/6 animals died during the observation period.

Both, the target substance C16-18 DMAPA amidoamine and the source substance Stearic acid 3-(dimethylaminopropyl)amide were not irritating to skin in an in vitro skin irritation test.

Both, the target substance C16-18 DMAPA amidoamine and the source substance Stearic acid 3-(dimethylaminopropyl)amide were not mutagenic in the bacterial reverse mutation assay when tested up to cytotoxic concentrations.

Also DMAPA did not show any alerts for genotoxic potential: DMAPA was not mutagenic in the Ames test and did not induce micronuclei in normochromatic erythrocytes from mouse bone marrow cells(NTP; OECD SIDS). All other study reports on genotoxicity published in the ECHA registered substance database did not raise any concern for genotoxic properties of DMAPA.

 

4.2 Quality of the experimental data of the analogues:

Genotoxicity:

The target substance C16-18 DMAPA amidoaminehas been tested in a reliable OECD TG 471 test.

The source substance Stearic acid 3-(dimethylaminopropyl)amide has been tested in reliable OECD TG 471, OECD TG 476 and OECD TG 473 test. All tests have been conducted according to GLP criteria. Therefore this data have no uncertainties and can be used in an analogue approach.

A reliable with restrictions, non-GLP study equivalent to OECD TG 471 is available for DMAPA, which is also sufficiently reliable.

 

4.4 Classification and labelling

Concerning human health hazards, the source substance Stearic acid 3-(dimethylaminopropyl)amide is classified for irreversible effects on the eye (Eye Damage, Category 1, H318: Causes serious eye damage. / Xi; R41 Risk of serious damage to eyes). Based on the read-across, the target substance C16-18 DMAPA amidoamine will be classified accordingly.

 

 

5. Conclusion

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles.

The negative outcome of the complete testing battery of in vitro genotoxicity tests for the source substance Stearic acid 3-(dimethylaminopropyl)amide is considered to be relevant also for the target substance C16-18 DMAPA amidoamine. This is further supported by studies suggesting no genotoxic potential for DMAPA. Thus, no classification for genotoxic properties is required.

 

 

References

NTP (National Toxicology Program),3-Dimethylaminopropylamine109-55-7; available online: http://ntp-server.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/Dimethylaminopropylamine_508.pdf

 

OECD SIDS,3-AMINOPROPYLDIMETHYLAMINE CAS N°: 109-55-7, available online: http://www.inchem.org/documents/sids/sids/109557.pdf

Justification for selection of genetic toxicity endpoint
No single key study has been selected since all available studies were negative.

Short description of key information:
- negative: Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (OECD TG 471 and GLP); cytotoxicity was observed at a concentration of >/= 10 µg/plate for the S. typhimurium strains and at >/= 1000 µg/plate for E. coli.
- negative: Mammalian cell gene mutation assay with mouse lymphoma L5178Y cells (TK) (met. act.: with andwithout) (OECD Guideline 476 and GLP); cytotoxicity was observed at concentrations of > 4 µg/mL without S9 mix and > 50 µg/mL with S9. (read across from Stearic acid 3-(dimethylaminopropyl)amide)
- negative: In vitro mammalian chromosome aberration test with cultured peripheral human lymphocytes (met. act.: with andwithout) (OECD Guideline 473 and GLP); cytotoxicity: no (but tested up to precipitating concentrations) (read across from Stearic acid 3-(dimethylaminopropyl)amide)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on reliable, relevant and adequate data on the target substanceC16-18 DMAPA amidoamine as well asfrom the closely related source substanceStearic acid 3-(dimethylaminopropyl)amide, the target substanceC16-18 DMAPA amidoamine is considered to be not mutagenic and not clastogenic.According to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008 no classification and labelling for mutagenicity is necessary.