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EC number: 246-807-3 | CAS number: 25307-17-9
In this report the in vitro rate as determined in more than 40 biotransformation tests is collated. The results of the first explorative tests were not included. A further refinement in the test setup was implemented based on the results observed for the C16 and C18 alkyl diethanolamines. A parallel sample setup was introduced to limit the effect of sorption to glassware.
Testosterone was 13 times included in tests as reference (benchmarking) substance. In the first tests also controls without S9 were included but these results were identical to the results observed with the inactive S9 and were for practical reasons not further included.
In Figure 2 the mean of 11 depletion curves (incl. SD) for both the active S9 and control are presented. To be able to present the results in one graph, all values were normalized to the mean initial value because slightly different starting concentrations (ranging from 10.05 – 11.89 µM) were used.
The results were fitted using linear regression and the clearance rate is derived from the slope of the regression line. The red line is the mean curve for the biotransformation results with the active S9 fraction. The blue line is the mean curve for the biotransformation results with the control (without S9).
The mean half-life (active minus control) for the 11 testosterone tests is 2.7 ± 0.34 hour. A summary of the testosterone data is presented in Annex 7. The relatively large standard deviation observed originates from the fact that these tests were performed over a period of 1½ year by different technicians who used two different batches of S9.
The mean half-life observed is about six times the half-life observed by Johanning et al (2010) of 0.46 h, who quantified the formation of 6ß-hydroxytestosterone at 500 µM, 12 °C using freshly prepared fish liver S9 indicating a potential lower activity of the commercially available S9 fraction used for these tests.
For each of the pure alkyl chain C12-C18 alkyldiethanolamines tests were performed at concentrations of 1 or 2.3 µM. Most tests were performed with the C12 alkyldiethanolamine for which the depletion results are presented in Annex 8. The results for the C14, 16 and 18 alkyldiethanolamines are presented in Annex 9. In Annex 10 the adequate mean depletion curves are presented for each alkyldiethanolamine. Like for testosterone the mean curves are presented after normalization to the initial concentration. The observed biotransformation rate of these mean curves is for each of the 4 tested alkyldiethanolamines together with number of tests, half-lives and standard deviation presented in Table 2:
Table 2: Observed mean biotransformation rate and half-life of alkyldiethanolamines
Alkyl chain length
Number of tests
Slope* of log transformed substrate depletion curve
t½ (h) measured
t½ (h) BCFBAF**
-0.119 ± 0.034
-0.103 ± 0.017
-0.051 ± 0.012
-0.011 ± 0.005
* The biotransformation rates are presented as negative values as they represent disappearance of the test substance
** normalized to 10 g fish
In the last column of Table 2 the half-life predictions are included as calculated by the BCFBAF model (EPIsuite 2012). From the results in Table 2 it is evident that the depletion rate decreases with an increasing alkyl chain length and that the BCFBAF model predicts a similar trend. A reduced bioavailability in the test is considered to be the most likely reason for this phenomenon.
Comparison of testing pure alkyl chain substance and technical mixtures
In addition to the pure alkyl chain diethanolamines also tests with the technical mixtures coco- and hydrogenated tallow diethanolamines were performed. The technical mixtures were like the pure alkyl chain diethanolamines tested at 1 and 2.3 µM test substance. The concentration of the two main constituents i.e. the C12 and C14 fraction of the coco diethanolamine were therefore respectively 52 and 18% of these nominal test substance concentrations. For hydrogenate tallow diethanolamine the two main constituents monitored for biotransformation were the C18 and C16 fraction which are respectively for 65 and 31% present in the test substance.
To evaluate whether there is a difference between results obtained with the pure alkyl chain diethanolamines and the technical mixtures, the depletion curves of the main constituent of each of the technical products were compared with the mean depletion curves of the pure alkyl chain diethanolamines. The results of this evaluation are presented in Figure 3.
In vitro to in vivo extrapolation for predicting the BCF
The BCF was predicted based on the extrapolation model (Version Public_062713)as described by Nichols et al. (2013). The results of these calculations are presented in Table 3 below. More detailed information on the model, parameters used and an example calculation (with hexadecyl diethanolamine) are included in Annex 12. Cationic surfactantsare amphipathic substances which partition mainly by other mechanisms than partitioning to lipids and consequently, BCF values for cationic surfactants should not be lipid normalized.
Table 3: Predicted BCF for C12-18 diethanolamine based onin vitrotoin vivoextrapolation.
using OECD 123*
Slope of log transformed substrate depletion curve
Estimated Partitioning based BCF(L/kg)
BCF with biotransformation (L/kg)
*Paulson, 2010a, b and c
** Negative values represent the decrease of the test substance
The observed rapid biotransformation of the C12to C18 alkyldiethanol amines demonstrates that it will be very unlikely that these substances will accumulate in fish. This was confirmed by the calculated BCF values which where were all below the CLP threshold value of 500 L/kg. It is therefore concluded that C12to C18alkyldiethanolamines have a low bioaccumulation potential and that anin vivoevaluation of the bioaccumulation potential by e.g. performing an OECD 305 bioaccumulation test is not expected to result in BCF values > 500 L/kg.
The measured log Kow values for C12to C18alkyldiethanolamines range from 1.5 to 4.4 (23 °C, 5<pH<6, Paulson, 2010) using the slow stirring approach according to OECD 123. A log Kow>3 indicates that a substance may have a bioaccumulation potential if the substanceisa narcotic substance. The observed rapid biotransformation of the C12to C18alkyldiethanol amines demonstrates that it will be very unlikely that these substances will accumulate in fish. This was confirmed by the calculated BCF values which where were all below the CLP threshold value of 500 L/kg. It is therefore concluded that C12to C18 alkyldiethanolamines have a low bioaccumulation potential and that an in vivo evaluation of the bioaccumulation potential by e.g. performing an OECD 305 bioaccumulation test is not expected to result in BCF values > 500 L/kg.
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