3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is highly insoluble in water, hence indicating that aquatic toxicity is unlikely to occur

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VII column 1, 9.1.1. Short-term toxicity testing on invertebrates (preferred species Daphnia) The registrant may consider long-term toxicity testing instead of short-term. According to column 2, 9.1.1. 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, or
— a long-term aquatic toxicity study on invertebrates is available, or
— adequate information for environmental classification and labelling is available.
The long-term aquatic toxicity study on Daphnia (Annex IX, section 9.1.5) shall be considered if the substance is poorly water soluble.

In general, there is no legal limit value available defining a substance as poorly water soluble or insoluble under REACH. However, in ECHA’s Guidance document Chapter R.7b: Endpoint specific guidance Version 3.0 – February 2016, it is stated: „poorly water soluble substances (e.g. water solubility below 1 mg/L or below the detection limit of the analytical method of the test substance)“. Further, poorly soluble substances are defined by OECD (2000 OECD SERIES ON TESTING AND ASSESSMENT, Number 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, ENV/JM/MONO(2000)6) as substances with a limit of solubility <100 mg/l although technical problems are more likely to occur at <1mg/l as defined in TGD (1996). Very low water solubility (i.e. in the low μg/l range) could be used as a reason to significantly modify a standard test or to test non-pelagic organisms preferentially.
ECHA’s Guidance document Chapter R.7c: Endpoint specific guidance Version 3.0 – June 2017 further says: „As indicated in the OECD TG 305, for strongly hydrophobic substances (log Kow > 5 and a water solubility below ~ 0.01-0.1 mg/L), testing via aqueous exposure may become increasingly difficult. However, an aqueous exposure test is preferred for substances that have a high log Kow but still appreciable water solubility with respect to the sensitivity of available analytical techniques, and for which the maintenance of the aqueous concentration as well as the analysis of these concentrations do not pose any constraints.

Prior to ecotoxicity studies a validation of an analytical method using LC MS MS for the determination of 3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide in the matrices demineralised water, algal and daphnia test medium in accordance with SANCO/3029/99 rev. 4 has been conducted:

Validation was planned in the concentration range 0.1 – 10 µg/L, initially. As no reliable calibration curve in this concentration range was obtained, but the peak area size was promising, the concentration range was lowered to 0.01 – 1 µg/L. In this concentration range, too, the measurement values in the calibration curves were scattering. Therefore a more sensitive analytical instrument was tested in the same concentration range, but with the same result of scattering measurement values.
As the test item is an ionisable substance, a buffering agent (ammonium formate) was added to the calibration solvent to prevent irreproducible interaction of the test item with glassware, caused by pH fluctuations. The use of ammonium formate did not improve the obtained results, therefore a second buffering agent (glycine at pH 10.5) was tested, but again without improvement.
It was concluded that measurements in the concentration range below 1 µg/L are not possible with sufficient reliability even though the absolute signal intensity is strong enough for sensitive measurements. The calibration range was changed to 1 – 50 µg/L for further experiments. After first evaluation of the calibration curve it was found that the range was chosen too wide. After restriction of the concentration range to 1 – 10 µg/L all validation criteria were fulfilled.
Solubility in matrices demineralised water, Algal test medium and Daphnia test medium was low. Peak areas, if detectable, were at least an order of magnitude smaller than the peak area of calibration standard 1 µg/L, independent of the filtration material used. Therefore testing of accuracy and stability of the test item in the matrices was not possible (spiking is not possible at concentrations higher than the solubility).
Although the method is considered suitable for the determination of the test item in calibration solvent, the determination of exact concentrations in matrices is not possible and the usability is limited to determine a cut-off concentration of 1 µg/L in matrices.

Based on the available information it can be stated that, as outlined in the first waiving possibility under REACH, the substance is highly insoluble in water, and hence, aquatic toxicity is unlikely to occur.

With a water solubility of < 2.59 µg/L the substance does not only need to be regarded as poorly soluble but practically insoluble in water. Hence, long-term testing does also not need to be conducted, as very low water solubility can be assumed if the substance is soluble only in the low μg/L range, and the actual water solubility is way below. Further, the registrant demonstrated that the analysis of concentrations in water poses constraints, the sensitivity of available analytical techniques is not sufficient to analyse concentrations in the estimated water solubility range.

In consequence, testing can be omitted.