1,3-diethyl-2,4-diisocyanato-5-methylbenzene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex XI, 2. TESTING IS TECHNICALLY NOT POSSIBLE, Testing for a specific endpoint may be omitted, if it is technically not possible to conduct the study as a consequence of the properties of the substance: e.g. very volatile, highly reactive or unstable substances cannot be used, mixing of the substance with water may cause danger of fire or explosion or the radio-labelling of the substance required in certain studies may not be possible. The guidance given in the test methods referred to in Article 13(3), more specifically on the technical limitations of a specific method, shall always be respected.
As depicted in more detail in the attached Expert Statement on the reactions of Isocyanates and Di-isocyanates (e.g. DETDI) with water, Isocyanates have the possibility to react under different pH-conditions in water.
Under neutral and acidic conditions first formation of unstable carbamic acid takes place. This decomposes immediately to the corresponding amine (neutral conditions) or ammonium salt (acidic conditions). Acidic conditions (catalysis) accelerate the formation of unstable carbamic acid.
Under basic conditions formation of carbamic acid anion should occur which does not decompose to the amine and carbon dioxide because no proton is available. The carbamic acid anion should be stable under basic conditions.
Isocyanates are capable to react with water via carbamic acid to the corresponding amine and carbon dioxide, the resulting amines are capable to react with isocyanates to form urea-derivatives. As isocyanates bear two isocyanate-groups, these molecules have the possibility to react further with water to form polyurea.
This reaction leads in the end to polymeric molecules, and the anticipated polyuria derivatives are not soluble in water and expected to have a high molecular weight. Due to the high molecular weight, a second waiving criterion is also met. According to REACH Annex VII column 2, the study does not need to be conducted if there are mitigating factors indicating that aquatic toxicity is unlikely to occur for instance if the substance is highly insoluble in water or the substance is unlikely to cross biological membranes, which is the case here. The reaction products are insoluble in water and too large, most probably way above 500 Da, to cross biological membranes.
Further, experiments were performed by an external laboratory (attached) to evaluate also practically the feasibility of ecotoxicity testing. As proposed above using common literature data, the pre-test for Ecotoxicity testing show that DETDI is rapidly transformed into another substance. In detail, the test item DETDI (3,5-Diethyltoluene-2,4-diisocyanate) is expected to be instable in water. The cyanide groups are supposed to react in aquatic environment to the respective amines. These amines are supposed to react with the residual cyanides to insoluble polymers. The half-life time of DETDI as well as the reaction products are not known.
Preliminary experiments in order to reveal information about the solubility of DETDI in test water (i.e. Elendt-Medium) were performed.
100 mg DETDI were added to 1 L test water and stirred for several times. Three applications were performed with stirring times of 10 minutes, 2 hours and 24 hours. After the respective time the applications were allowed to settle down for 15 minutes in order to separate the water accommodated fraction. No undissolved particles or micelles were determined by optical inspection of the middle phase from which 2 samples of 20 mL were drawn.
The samples were extracted twice with 5 mL Toluol for 10 minutes each. The extracts were merged and analysed by GC-MS. The recoveries were as follows:
10 minutes stirring: about 1% = 1 mg/L of the application of 100 mg/L
2 hours stirring: about 8% in one replicate and about 14% in the second replicate
= 8 and 14 mg/L of the application of 100 mg/L
24 hours stirring: about 3 % = 3 mg/L of the application of 100 mg/L

Based on these results the range-finder pre-test was performed.
The stirring time of the 100 mg DETDI/L solution was 2 hours. The separation time for the range-finder test was adapted to 1 hour. This was done because it was assumed that the separation time was not sufficient, which is indicated by the difference between the replicates of the 2 hours stirring time of the solubility pre-test.
This solution was used as test medium sample and to prepare the test medium samples of dilutions of 1:10, 1:100 and 1:1000. These solutions were applied to the test system.
The samples of test start and test end were drawn and extracted in the same way as described above.
In order to validate the analytical method according to SANCO/3029/99 fortification level of 0.08 and 10 mg DETDI/L were prepared at test start and test end. Therefore, a stock solution of 1 g DETDI/L in acetonitrile was prepared. Appropriate amounts of this solution were diluted further with test water to obtain the fortification level of 10 mg/L. Intermediate dilutions of 100 mg DETDI/L in acetonitrile and 1 mg DETDI/L in test water were used to obtain the fortification level of 0.08 mg/L. The extraction was performed as described above.
The extracts of the fortification samples and biological samples of test start were stored in the refrigerator at 4±4°C until analysis was performed at test end. The extracts of the samples of test end were prepared directly after sampling and analysed immediately.
The high fortification level and the biological samples of the nominal concentrations of 100 and 10 mg DETDI/L were diluted by a factor of 100 with Toluol to match the calibration range. The biological samples of the nominal concentrations of 1 and 0.1 mg DETDI/L were diluted by a factor of 10 with Toluol.
The recoveries of the fortification samples should be between 70 and 110 % of its nominal concentration. The recoveries of the fortification level were as follows.
Low fortification level: 10 % in the samples prepared at test end, 1 % in the samples prepared at test start.
High fortification level: 40 % in the samples prepared at test end, 12-15 % in the samples prepared at test start.
Those results indicate that the samples are not stable during storage in the refrigerator. Moreover, either the extraction procedure is not sufficient or the reactivity of the test item with the test water in the fortification level is so quick that the extraction of the test item is not possible.
Reliable results of the biological samples were only obtained for the highest test concentration of nominal 100 mg DETDI/L. The recovery was approximately 80 µg/L in the sample of test start and below Limit of Detection in the sample of test end.
The OECD Series on Testing and Assessment No.23: “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures” suggests to test either the substance (parent compound) itself or its degradation products in case of hydrolysis. The procedure is dependent on the half-life time of the substance. If the half-life time is > 3 days one should test the parent compound. With a half-life time < 3 days and > 1 hour one should consider a case-by-case decision and include possible testing of degradation products. With a half-life time < 1 hour one should test the degradation products.
The pre-test for Ecotoxicity testing show that DETDI is rapidly transformed into another substance. However, the design of the pre-test does not allow determination of neither the half-life time nor the degradation products for DETDI due to the anticipated rapid polymerization of the hydrolysis products. Thus, the author concluded that testing DETDI in aquatic ecotoxicological studies would not result in reliable conclusions concerning possible effects during testing. As the anticipated polyurea derivatives are not soluble in water and expected to have a high molecular weight, aquatic ecotoxicological studies will not have any reliable outcome.