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

Endpoint summary

Additional information

No experimental aquatic toxicity data are available for C16-18 DMAPA amidoamine. However, studies with the read-across substance Stearic acid 3-(dimethylaminopropyl)amide are available.

The test item has a low water solubility and sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organism and walls of the test vessel) and results from such tests depend from the test settings applied. Using natural river water which contains particulate as well as dissolved organic carbon to which the test item can sorb partially reduces the difficulties encountered in tests with synthetic water e.g. preventing that the test item settles onto surfaces. The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries. Due to the short exposure period these low recoveries cannot be associated to biodegradation. This means the test substance is present in the test system and therefore available for exposure (dissolved in water and sorbed also called bulk). This so called Bulk Approach is described by ECETOC (2003).

A justification for read-across is given below.

Short-term toxicity to fish

In an acute fish test with rainbow trout no mortality was observed at the limit test concentration of 0.1 mg/L. In a range finding test also at 0.1 mg/L no mortality was observed but at 1 mg/L 100% mortality occurred. This means the LC50 (96h) is in the range of 0.1 and 1 mg/L. This result is regarded as sufficient as a chronic fish test result with a NOEC of 0.1 mg/L is available.

Long-term toxicity to fish

The effects of the test item to the embryo and sac-fry stages of the zebrafish (Danio rerio) were determined according to OECD Guideline 212. A semi-static test procedure with natural river water and daily renewal of the test media was performed with the nominal test item concentrations of 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L.

The test was started by placing fertilized eggs in the test vessels and lasted 9 days (5 days post-hatch). 30 eggs of Danio rerio were exposed per test concentration and control (3 replicates with 10 eggs each), respectively. On day four 93 % of the control larvae have hatched. Therefore, study day 4 was defined as post hatch day 0 (PHD 0).

Different toxic endpoints were determined: egg hatch, time to hatch, post hatch survival, overall fry survival and mortality, fry growth (expressed as length and weight), morphological and behavioural effects. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and LC-values were determined based on the statistical results.

All concentrations of the test item and the control were analytically verified by LC-MS/MS at the start and the end of three exposure intervals.

 

NOEC, LOEC: Hatch, Fry Survival, Growth, Behaviourbased on nominal test item concentrations of the read-across substance Stearic acid 3-(dimethylaminopropyl) amide

Parameter	NOEC [mg/L]	LOEC [mg/L]
Hatch	1.00	3.16
Post hatch survival	0.316	1.00
Overall survival	0.316	1.00
Length	0.316	1.00
Weight	0.316	1.00
Abnormal behaviour (Quiescence, Remaining unusually long at the bottom)	0.100	0.316

 

Short-term toxicity to aquatic invertebrates

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item Stearic acid 3-(dimethylaminopropyl)amide were determined according to OECD 202 (2004).

The study was conducted under semi-static conditions over a period of 48 h with 5 concentrations of the test item Stearic acid 3-(dimethylaminopropyl)amide in the range of 62.5 to 1000 µg/L, prepared with natural river water in a geometric series with a separation factor of 2. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the test item Stearic acid 3-(dimethylaminopropyl)amidewereanalytically verified by LC-MS/MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control.

The 48 h EC50 was381 µg/L (95% confidence interval 321 – 427 µg/L)(based on the nominal concentrations).

Long-term toxicity to aquatic invertebrates

The Daphnia magna Reproduction Test (semi-static, 21 d) of the test item Stearic acid 3-(dimethylaminopropyl)amide was conducted according to OECD 211 (2008). Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level, and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with a daily renewal of the test solutions. Nominal concentrations of the test item were selected as follows: 25.0–50.0–100–200–400 µg/L. The concentrations of the test item were analytically verified by LC-MS/MS of samples taken at the start of the exposure intervals on days 0, 2, 8, 15 (0 h) and at the end of the exposure intervals on days 1, 3, 9, 16 (24 h) of all concentration levels and the control.

The adult mortality was the only significant effect in this study. Effects concerning the reproduction were not observed during the test period of 21 days.

Based on the significant adult mortality of 100 % at the concentration level of 400 µg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 200 µg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 400 µg/L.

The EC10 for the adult mortality was calculated by sigmoidal dose-response regression to be 200 µg/L (95% confidence limits: 100 – 400 µg/L). The EC50 was calculated accordingly to be 212 µg/L (95 % confidence limits: 100 – 400 µg/L). Effect levels are given as nominal concentration.

Toxicity to aquatic algae and cyanobacteria

The toxicity of Stearic acid 3-(dimethylaminopropyl)amide to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006).The aim of the study was to assess the effects on growth rate and yield in natural river water over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 4332 cells/mL.Based on a preliminary test, 5 concentration levels were tested in a geometrical series with a dilution factor of nominal: 10.0 - 31.6 - 100 - 316 - 1000 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

All test concentrations of the test item and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 h) and the end of the exposure (72 h).
In this study the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 µg/L. The LOEC-values for both inhibition of growth rate and yield after 72 hours were 100 µg/L. The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 140 (130 – 154) µg/L. The EC50-value for inhibition of yield (EyC50) with 95% confidence intervals after 72 hours was 74.3 (68.1 – 82.0) µg/L. All effect levels are given based on nominal concentrations.

Toxicity to microorganisms

In an OECD 209 Sludge respiration inhibition test no effect was observed at the limit test concentration of 100 mg/L. Based on the range finding test it can be estimated that the EC50 (3h) is between 100 and 1000 mg/L. The 3 h NOEC was 100 mg/L.

1. Read-across hypothesis and justification

This read-across is based on the hypothesis that source and target substances have similar ecotoxicological 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).

·        of their structural similarities: target and source substances are comprised of a hydrophobic (alkyl) and hydrophilic (positively charged ammonium) part; due to theses structural elements they form micelles and have surface active properties.

·        of their similar molecular weight, physicochemical properties and similar ecotoxicological profile in aquatic tests

Therefore, read-across from the existing ecotoxicological studies on the source substances is considered as an appropriate adaptation to the standard information requirements of Annex VII 9.1.1, 9.1.2, 9.2.1, Annex VIII 9.1.3, 9.1.4, Annex IX 9.1.5, 9.1.6, 9.2.1, and 9.4 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 substances		Target substance
	Stearic acid 3-(dimethylaminopropyl)amide	N,N,N-trimethyl-C20-22-(even numbered)-alkyl-1-aminium chloride; C20/22- alkyltrimethylammonium chloride (C20/22-ATQ)	C16-18 DMAPA amidoamine
Substance type	mono constituent substance	UVCB	UVCB
CAS number	7651-02-7	68607-24-9
EC number	231-609-1	271-756-9
Chain length distribution	< C16: < 1.6% C16: < 7% C18: > 89.8% > C18: < 1.6%	C16: <1% C18: ca. 4% C20: ca. 12% C22: ca. 82% C24: ca. 1%	C14: <= 5 % C16: 25-35 % C18: >= 61 %
DMAPA	<0.002%	N/A	<=0.01%

 

2.1 Substance Identity

Substance descriptions

The target substance C16-18 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 substance Stearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid and N, N-dimethylpropylenediamine. It is composed of mainly C18 amides (> 89.8%) of DMAPA and small amounts of the C16 amide (<7%).

The source substance C20/22-ATQ is manufactured from behenyl alcohol and dimethylamine, resulting in the corresponding tertiary amine dimethylalkylamine. In step two, the dimethylalkylamine is treated with methylchloride at elevated pressure to form the quaternary ammonium chloride.

2.2 Common breakdown products

The source substance Stearic acid 3-(dimethylaminopropyl)amide is the main component of the UVCB target substance C16-18 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. 

As no ecotoxicological data are available for the target substance C16-18 DMAPA amidoamine, but only its main constituent Stearic acid 3-(dimethylaminopropyl)amide, comparison of ecotoxicological data of the second source substance C20/22 ATQ is only possible to the latter. However, as explained above, the small amount of additional C16 is not expected to have any relevant influence on toxicity.

Both, target and source substances are cationic surfactants and are strongly sorbing to solids due to ionic interactions and van der Waals forces. Both substances are also readily and ultimately biodegradable in an OECD 301B CO2 Evolution test. In addition the microbial metabolic pathway is the same for both substances as in the first step the alkyl chain is cleaved from the nitrogen forming the corresponding aldehyde and ammonium compound. The aldehyde is then oxidised to the fatty acid which is subsequently degraded by beta oxidation (Kees van Ginkel, Handbook of Surfactants, Volume F, 1995).

2.3 Common structural elements

Target and source substances are comprised of a hydrophobic (alkyl) and hydrophilic (positively charged ammonium) part. Due to theses structural elements they form micelles and have surface active properties.

2.4 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 ecotoxicity.

C16-18 DMAPA amidoamine as well as Stearic acid 3-(dimethylaminopropyl)amide are protonated to a large degree at environmentally relevant pH. Whereas C20/22 ATQ is a quaternary ammonium chloride. This difference is not considered to be of ecotoxicological relevance.

C16-18 DMAPA amidoamine as well as Stearic acid 3-(dimethylaminopropyl)amide on the one side contain DMAPA as amine-backbone; C20/22 ATQ on the other side is based on dimethylamine as amine-backbone. However, as it is shown in the aquatic toxicity tests with Stearic acid 3-(dimethylaminopropyl)amide and C20/22 ATQ, this has no big influence on ecotoxicity.

 

3. Physicochemical properties:

Table 2: Physicochemical properties

	Source substances		Target substance
Endpoints	Stearic acid 3-(dimethylaminopropyl) amide	C20/22-ATQ	C16-18 DMAPA amidoamine
Molecular weight [g/mol]	368.64	ca. 400	340.59 - 368.64
Physical state at 20°C / 1013 hPa	Solid (paste)	Solid	Solid (waxy)
Melting point	67.4°C	Decomposition at 220-240°C	41.8°C
Boiling point	412.3°C	Decomposition	320.5°C
Surface tension	37.86 mN/m at 0.22 g/L	47.0 mN/m at 0.01 g/L	26.7 mN/m at 0.027 g/L
Water solubility	10 mg/L at 20°C	10 mg/L at 25°C	3.65 mg/L at 23°C
Log Kow	2.01 at 20°C, pH7	3.29 at 20°C	2.01 at 20°C, pH7; read-across fromStearic acid 3-(dimethylaminopropyl) amide
Vapour pressure	3.4E-08 Pa at 20°C	7E-05 Pa at 20°C	read-across from   Stearic acid 3-(dimethylaminopropyl) amide
Adsorption / desorption	no data (read-across from C20/22-ATQ)	log Koc = 3 – 5.7 (batch equilibrium method)	log Koc = 7.8-8.0 at 25°C (HPLC method)

 

As demonstrated in the table above, the source substances have a similar physicochemical profile compared to the target substance. The substances are cationic surfactants and are strongly sorbing to solids due to ionic interactions and van der Waals forces.

4. Comparison of data from ecotoxicological endpoints

5.1 Ecotoxicity data of the target and source substances

Cationic surfactants sorb strongly to negatively charged surfaces like glass or biota. In order to avoid sorption to the glass of the test vessel and on the test organism well characterised river was used as aquatic medium to allow reliable test results.

 

Table 4: Ecotoxicological profiles for the source substances Stearic acid 3-(dimethylaminopropyl) amide and C20/22-ATQ, and the target substance C16-18 DMAPA amidoamine

	Source substances		Target substance
Endpoints	Stearic acid 3-(dimethylaminopropyl) amide	C20/22-ATQ	C16-18 DMAPA amidoamine
Short-term toxicity to fish	96 h LC50 >0.1 - <1 mg/L(nominal)	96 h LC50 = 3.5 mg/L (meas., geom.. mean)	No data; read-across
Long-term toxicity to fish	9 d NOEC(behaviour) = 0.1 mg/L (nominal)	9 d NOEC = 0.24 mg/L (meas., geom.. mean)	No data; read-across
Short-term toxicity to aquatic invertebrates	48 h EC50 = 381 µg/L (nominal)	48 h EC50 = 1.39 mg/L (nominal)	No data; read-across
Long-term toxicity to aquatic invertebrates	21 d NOEC(mortality) = 200 µg/L (nominal); 21 d EC10 (mortality) =200 µg/L (nominal)	21 d NOEC = 128 µg/L	No data; read-across
Toxicity to aquatic algae and cyanobacteria	72 h EC50 = 140 µg/L (nominal); 72 h EC10 = 71 µg/L (nominal)	72 h EC50 = 3.48 mg/L (meas., geom. mean); 72 h EC10 = 0.78 mg/L (meas., geom. mean)	No data; read-across
Toxicity to microorganisms	3 h EC50 >100 - < 1000 mg/L; 3 h NOEC = 100 mg/L	3 h EC50 >100 - < 1000 mg/L; 3 h NOEC = 100 mg/L	No data; read-across
Biodegradation in water; screening	readily biodegradable	readily biodegradable	readily biodegradable
Toxicity to soil macroorganisms except arthropods (earthworm)	No data	54 d NOEC(reproduction, mortality,body weight) = 250mg/kg soil dw   14 d NOEC(mortality, biomass, development) = 1000 mg/kg soil dw	No data; read-across
Toxicity to terrestrial arthropods (Collembola)	No data	28 d NOEC(mortality, reproduction) = 500 mg/kg soil dw;   28 d LD50 > 1000 mg/kg soil dw	No data; read-across
Toxicity to soil microorganisms	No data	28 d EC50 = 76 mg/kg soil dw; 28 d EC10 = 15 mg/kg soil dw; 28 d NOEC = 10 mg/kg soil dw	No data; read-across

 

No experimental ecotoxicity data are available for the target substance C16-18 DMAPA amidoamine. However, as demonstrated above,Stearic acid 3-(dimethylaminopropyl) amide is the main constituent of the target substance. Thus, aquatic toxicity data of the source substance Stearic acid 3-(dimethylaminopropyl) amide are considered to be relevant also for the target substance C16-18 DMAPA amidoamine.

Terrestrial ecotoxicity data are available for the source substance C20/22-ATQ. Based on the close similarity of Stearic acid 3-(dimethylaminopropyl) amide and the target substance C16-18 DMAPA amidoamine, also the read-across from C20/22-ATQ is justified:

The substances are readily and ultimately biodegradable. In addition the microbial metabolic pathway is the same for both substances as in the first step the alkyl chain is cleaved from the nitrogen forming the corresponding aldehyde and ammonium compound. The aldehyde is then oxidised to the fatty acid which is subsequently degraded by beta oxidation (Kees van Ginkel, Handbook of Surfactants, Volume F, 1995).

In the following table the river water test results for Stearic acid 3-(dimethylaminopropyl) amide and C20/22 ATQ are given.

		Stearic acid 3-(dimethylaminopropyl) amide	C20/22 ATQ	Ratio highest:lowest value
Chronic fish OECD 212 River water test	NOEC repro (9d)	0.1 mg/L	0.24 mg/L	2.4
Chronic daphnia OECD 211 River water test	NOEC repro (21d)	0.2 mg/L	0.13 mg/L	1.5
Algae OECD 201 River water	ErC10 (72h)	0.07 mg/L	0.93 mg/L	13.2
Algae OECD 201 Reconstituted water	ErC10 (72h)	0.2 mg/L	0.93 mg/L(RW)	4.7

 

The ecotoxicity ratios for Chronic fish and Chronic daphnia is 2.4 and 1.5 respectively. These ratios are well within the boundaries on variability / uncertainty accepted e.g. for reference substances. The ratio for ErC10 (72h) for Stearic acid 3-(dimethylaminopropyl) amide and C20/22 ATQ is 12.9 and much higher than expected. But from the tertiary alkyl dimethylamines (see above) it is known that the algae ecotoxicity in river water is sometimes higher than in reconstituted water. When comparing the Stearic acid 3-(dimethylaminopropyl) amide algae ErC10 (72h) in reconstituted water with the algae value for C20/22 ATQ river water the ratio for the endpoint drops from 12.9 to 4.5. The value 4.5 is well within the range for variability / uncertainty acceptable for algae test (3 standard deviations). The PNEC freshwater and marine for Stearic acid 3-(dimethylaminopropyl) amide is derived from the ErC10 (72h) river water algae test of 0.07 mg/L whereas the PNEC freshwater and marine for C20/22 ATQ is derived from the NOEC repro (21d) daphnia of 0.13 mg/L. The ratio between these two NOEC is 0.13/0.07=1.9 and well acceptable with respect to uncertainty / variability of test results.

Based on these data as well as on similar physicochemical properties and on the similar toxicological profile the read-across approach for long-term toxicity as well as terrestrial toxicity is considered to be appropriate. The available data are comparable for source and target substance, supporting the validity of the grouping approach.

5.3 Quality of the experimental data of the analogues:

Aquatic toxicity:

The source substance Stearic acid 3-(dimethylaminopropyl) amide has been tested in reliable (RL1) GLP-compliant studies according to OECD TG 201 and 202, and a reliable (RL2) study according to OECD TG 203 for short-term toxicity as well as reliable (RL1) GLP-compliant studies according to OECD TG 211 and 212 for long-term toxicity.

The source substance C20/22 ATQ has been tested in reliable (RL1) GLP-compliant studies according to OECD TG 201, 202 and 203 for short-term toxicity as well as reliable (RL1) GLP-compliant studies according to OECD TG 211 and 212 for long-term toxicity.

The available data from the source chemical are sufficiently reliable to justify the read-across approach.

5.4 Classification and labelling

Concerning environmental effects, the source substance Stearic acid 3-(dimethylaminopropyl) amide is classified as Aquatic Acute 1 (M-factor = 1) and Aquatic Chronic 2. The source substance C20/22 ATQ is classified as Aquatic Acute 1 (M-factor = 1).

Based on read-across, the target substance C16-18 DMAPA amidoamine is classified as Aquatic Acute 1 (M-factor = 1) and Aquatic Chronic 2.

The substances are neither PBT nor vP/vB substances.

6. Conclusion

The structural and physicochemical 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.

As demonstrated, Stearic acid 3-(dimethylaminopropyl) amide is the main constituent of the target substance C16-18 DMAPA amidoamine.

Based on close the relationship the results from aquatic, sediment and terrestrial toxicity data obtained with the source substances Stearic acid 3-(dimethylaminopropyl) amide and C20/22 ATQ are also relevant for the target substance C16-18 DMAPA amidoamine.