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EC number: 247-660-8 | CAS number: 26401-35-4
Mean Cumulative Offspring per Survivor in Daphnia magna after 21 d, as %Inhibition of mean cumulative offspring per survivor caused by the test item after 21.0 d.
Treatment Mean Std. Dev. n %Reduction
Control 94 12.8 9 0.0
1:10 dilution 92 14.8 10 1.2
eluate 69 16.7 10 26.7
The underlying study was performed to assess the long-term toxicity of diisotridecyl adipate to Daphnia magna. The test was conducted according to OECD guideline 211 under GLP.
Based on the available information prior to the test, indicating a water solubility of the test item of < 0.001 mg/L, the column elution method was chosen for preparing the test medium and obtaining the highest possible test concentration. For test medium preparation, a column was filled with column matrix loaded with the test item (1 mg/g loading rate, column material: silica gel 60). The top and the bottom of the fillings were covered with membrane filters to avoid the loss of matrix material. A constant flow of dilution water is pumped through the column from bottom to top with a flow rate of 10 ml/min. Purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a limestone column, and aeration.
Young female Daphnia, aged less than 24 hours at the start of the test (10 replicates of a single daphnid per group) were exposed to a steady state column eluate in form of the 100% eluate and 10 % eluate for a period of 21 days. The test solutions were renewed on a daily basis (working days, latest after 3 days; semi-static exposure). The treatment groups were compared to the control group in regard to the total number of living offspring produced per parent animal alive at the end of the test. Other substance-related effects on parameters such as growth (length), and possibly intrinsic rate of increase, are also examined. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The daphnids were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.
Analysis of the fresh and old test media (Day 1, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 18, 19, 20, 21) column eluate and the 10%-eluate, resulted in time weighted average (TWA) test item concentrations of 17.22 µg/l and 1.64 µg/l, respectively. Based on the test medium preparation method, it was considered justifiable to base the results on the mean measured test concentrations, as no nominal concentration is available. Both of the measured concentrations are above the water solubility reported for DITA.
No significant differences between the control group and the test groups were found for the following endpoints: Mobility, age at first reproduction, length and intrinsic rate. In the undiluted eluate, a ~27% reduction of mean cumulative offspring per surviving daphnids after 21 days were seen, whereas the exposure to the 10%-dilution of the eluate resulted in no significant effects for this endpoint, as well.
As mentioned above, the concentrations measured in the undiluted eluate are far above the reported and experimentally determined water solubility for diisotridecyl adipate. Hence, micelle formation or other processes causing the inhibition cannot be excluded.
Based on the results of this test the 21d NOEC value for mean cumulative offspring per survivor was determined to be 1.64 µg/L (based on the time weighted average (TWA) test concentration of the test medium), which is greater than the level of water solubility.
In a valid 21 day reproduction test according to OECD test guideline 202 (1984, part 2), the water flea Daphnia magna was exposed to three different concentrations of the read-across substance di-2 -ethylhexyl adipate (nominal concentrations 0.5, 1.0, and 2.0 mg/L, measured concentrations 0.19, 0.39, and 0.77 mg/L). To solubilise the test substance, a vehicle was used (1g/L MARLOWET R 40).
During the test, no effects were observed. Based on reproduction, a NOEC of>= 0.77 mg/L was determined. Toxic effects on reproduction and mortality of adult daphnids are above the water solubility of the test substance (Huels AG, 1996a).
The study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2008) No 211, “Daphnia magna Reproduction Test” referenced as Method C.20 of Commission regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1300 “Daphnid Chronic Toxicity Test”.
Information provided by the Sponsor indicated the water solubility of the test item to be less than 0.050 mg/L. Additionally, pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing . A pre-study media preparation trial indicated that the most appropriate method of preparation was using a solvent spike followed by filtration.
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of test concentrations of 10, 18, 32, 56 and 100% v/v solution for a period of 21 days. The test solutions were renewed 3 times per week. The test item solution was prepared by initially dissolving 100 mg of test item in a final volume of 10 mL of dimethylformamide. An aliquot of the solvent stock solution was dispersed in Elendt M7 media using a magnetic stirrer for approximately 10 minutes to give a 1.0 mg/L stock solution. After stirring, any undissolved test item was removed vial filtration to give the 100% v/v solution test concentration, from which dilutions were performed to give the remainder of the test series. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.
Analysis of the freshly prepared 100 % v/v solution preparation on days 0, 5, 12 and 19 showed measured concentrations to range from less than the limit of quantification to 0.0024 mg/L. Analysis of the old or expired media on days 2, 7, 14 and 21 showed measured concentrations to range from less than the limit of quantification to 0.00093 mg/L. Given the overall decline in measured concentration between each period of media renewal, it was considered justifiable to base the results on the mean measured test concentrations.
The “Lowest-Observed Effect Concentration” and the “No Observed Effect Concentration” based on the mean measured test concentration of the test media were greater than and equal to 0.00063 mg/L respectively. The “Maximum Acceptable Toxicant Concentration” was calculated to be 0.00063 mg/L. This study showed no toxic effects at the limit of water solubility.
NOEC(21d) = 1.64 µg/L (measured, column elution method) for immobilisation of Daphnia magna (OECD 211)
Chronic Daphnia data are available for DITA itself and the respective read across substances.
Key study / Diisotridecyl adipate
The available long-term toxicity study towards aquatic invertebrates for DITA (Simon, 2016) was conducted by using the column elution method for test medium preparation. Daphnids were exposed to a steady state column eluate in form of the 100% eluate and 10 % eluate for a period of 21 days in a semi-static test design. No significant differences between the control group and the test groups were found for the following endpoints: Mobility, age at first reproduction, length and intrinsic rate. In the undiluted eluate, a ~27% reduction of mean cumulative offspring per surviving daphnids after 21 days were seen, whereas the exposure to the 10%-dilution of the eluate resulted in no significant effects for this endpoint, as well.
The concentrations measured in both, the undiluted eluate and the 10%- eluate tests solution, are above the reported and experimentally determined (e.g. Kotthoff, 2016) water solubility for DITA of ~0.7 µg/L. Hence, micelle formation or other processes causing the inhibition cannot be excluded.
As seen for the read-across substances (details below) and based on the valid water solubility figure for DITA (0.7 µg/l), this study supports the fact that there is no toxicity in the range of water solubility.
Supporting information / Read-Across substances
In addition, there are three long-term toxicity tests with aquatic invertebrates available on the read-across substance bis(2-ethylhexyl) adipate (CAS 103-23-1; DEHA) and one study on the read-across substance diisotridecyl dodecanedioate (CAS 84731-63-5), which show no toxicity in the range of water solubility. All of these substances, DEHA, DITA and diisotridecyl dodecanedioate, are dialkylesters of dicarboxylic acids, either adipic acid (1,6-hexanedioic acid) or 1,12-dodecandioic acid. Both dicarboxylic acids are linear and have even-numbered carbon chains. The alcohol component of all three substances has branched alkyl groups (C8 or C13).
DEHA Chronic Daphnia studies showing non-toxicity
In a valid 21 day reproduction test according to OECD test guideline 202 (1984, part 2), the water flea Daphnia magna was exposed to three different concentrations of bis-(2-ethylhexyl) adipate (nominal concentrations 0.5, 1.0, and 2.0 mg/L, measured concentrations 0.19, 0.39, and 0.77 mg/L). To solubilise the test substance, a vehicle was used (1g/L MARLOWET R 40). During the test, no effects were observed. Based on the reproduction rate, a NOEC of >= 0.77 mg/L was determined (Huels AG, 1996a). This is considered a valid proof of the absence of long-term aquatic toxicity of bis(2-ethylhexyl) adipate to invertebrates in the range of water solubility. This result is confirmed in another study performed by Robillard et al. (2008) (key study). A chronic Daphnia magna limit test was conducted at an average exposure concentration of 4.4 μg/L (measured water solubility = 5.5 µg/L) in laboratory diluent water to avoid insoluble test material and physical entrapment. One hundred percent of the DEHA-treated organisms survived compared to 90% survival in both the controls and solvent controls. Mean neonate reproduction was 152, 137, and 148 and mean dry weight per surviving female was 0.804, 0.779, and 0.742 mg in the treatment, control and solvent control, respectively. No adverse effects were observed. In the third study (Felder et al. (1986), not conducted according to a current guideline and GLP), which is the only one with DEHA detailed discussed in the Substance Evaluation Draft Decision, a significantly reduced yield of young per adult per day at mean measured exposure levels of 0.087 and 0.18 mg/L were found. A MATC (maximum acceptable toxicant concentration) for long-term toxicity to Daphnia magna was calculated to be between 0.024 and 0.052 mg/L based on statistical analyses of adult mean length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate. Acetone was used as a solvent in this study. No adverse effects to survival growth or reproduction were observed even at levels five times the water solubility of DEHA. Effects were reported at concentrations significantly higher than the water solubility of DEHA (0.005 mg/L). Under such conditions it is likely that the observed effects have been caused by physical entrapment rather than chemical interactions (Rhodes et al. 1995). Furthermore, information provided in the results section of the publication is rather scarce. Neither raw data nor details about the extent of impairment daphnids had to face at a given concentration are given, and no information about a dose-response relationship is included. In our opinion also the study of Felder et al. (1986) with the NOEC of 0.024 mg/L shows that no toxicity occurred within the range of water solubility of 0.005-0.0032 mg/L. The lowest measured water solubility value of DEHA is lower than the NOEC. The study supports the fact that there was no toxicity in the range of water solubility. Therefore this value cannot be used for the PNEC-derivation.
Diisotridecyl dodecanedioate Chronic Daphnia study showing non-toxicity
In addition to the presented long-term toxicity studies with Daphnia magna performed with DEHA, a recently (2013) conducted long-term toxicity test with Daphnia magna (OECD 211) on the read-across substance diisotridecyl dodecanedioate is available. The GLP study was performed under semi-static conditions using DMF as a solvent (key study). In the Substance Evaluation Draft Decision it is discussed by the MSCA that the study cannot be considered valid due to uncertainties in the preparation of samples which resulted in declining of 99 % of the test substance in 48 h to below the LOQ. We would like to defend the study and attached a letter with the complete rationale of the responsible test institute Harlan. "The decline in measured test concentrations in the inoculated test samples was due to adsorption of the test item to the algal cells that were present. In the Daphnia magna Reproduction Test, algal cells are added to the test solutions in order to provide a food source for the daphnids, it is therefore considered that the decline in measured concentrations over each test media renewal period in this test was due to adsorption of the test item to the algal cells and not due to instability and / or volatility. As the test item was adsorbed to the algal cells that the daphnids ingest as a food source it can be considered that the daphnids were exposed to the test item over the period of each test media renewal" (please see attached the complete wording of the rationale). Furthermore based on the structure of diisotridecyl dodecanedioate it is expected that this substance is even more water insoluble than DITA (< 0.001 mg/L).The analytics for such water insoluble UVCB-substances are very difficult. The measured concentrations in this study were extremely low. Therefore it is not unlikely that the measured concentrations were inconsistent in such a sensitive study. Analysis of the freshly prepared 100 % v/v solution preparation on days 0, 5, 12, 19 showed measured concentrations ranging from less than the LOQ (assessed as 0.00027 mg/L) to 0.0024 mg/L. Analyses of the old or expired media on days 2, 7, 14, 21 showed measured concentrations ranging from less than the limit of quantification to 0.00093 mg/L. Given to the apparent decline in measured concentration between each period of media renewal, it was considered justifiable to base the results on the mean measured test concentrations of the test media to give a "worst case" analysis of the data. The No Observed Effect Concentration based on the mean measured concentration of the test media was equal to 0.00063 mg/L respectively. The mean measured concentration of 0.00063 mg/L was estimated for the 100 % v/v solution. We provide additional information including a better justification for the choice of test solution preparation used for the diisotridecyl dodecanoate test (Harlan 2013), and why it would achieve saturation for a low solubility UVCB (please see attachment “issue1_OECD211comments2_diisotridecyldodecanoate_studyno41202662.pdf"). In the study of Robillard et al. (2008) the water solubility of 5.5 (± 0.22) μg/L for di(2- ethylhexyl) adipate (DEHA) was measured using the slow-stir method which is in the Registrants’ scientific opinion, one of the most suitable methods for substances with a very low water solubility. Compared to DEHA, the water solubility of DITA and of diisotridecyl dodecanoate can be expected to be lower because of longer C-chains. Therefore, the higher measured water solubility values for DITA and diisotridecyl dodecanoate must be interpreted with caution, since they were determined with the ASTM-E1148 Standard Test Method, a comparably less sensitive method. In general, the determination of a definite value for the water solubility of UVCB substances poses a very complex problem and is by far more complicated than the determination for single substances (monoconstituents). This study shows like the DEHA-studies no toxic effects at the limit of water solubility.
Overall assessment of Chronic Daphnia for DITA
Thus, on the basis of the available data for DITA and the supporting information, it can be concluded that DITA is neither acutely, nor chronically toxic to aquatic invertebrates up to its limit of water solubility.
Although the measured water solubility values for DITA, as mentioned above, are lower than the determined NOEC in this study, the obtained NOEC (Simon, 2016) will be used as basis for the PNEC-derivation and hence be taken into account for the environmental exposure assessment.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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