A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

Endpoint summary

Description of key information

Bioaccumulation in aquatic species is based on an experimental BCF test according to OECD TG 305 with aqueous exposure and result in a BCFssl of 670 l/kg using the highest BCF of one of the four constituent and the higher BCFssl compared to BCFkl. 

Bioaccumulation in terrestrial species is based on the available information (log Kow). The BCFs for terrestrial organisms were calculated using QSARs of Jager (1998) incorporated in the EUSES model, and yielded value 631 L/kg ww, respectively, using a log Kow of 4.72.

Bioaccumulation for air-breathing organism is based on available information of Log Koa, persistency information, identification of the key degradation product and anticipated metabolism. The Koa for Cassiffix exceeds the cut off criterion of 5. The PBT working group considered Cassiffix P based on OECD TG 309. In this biodegradation study Cassiffix-Lactone has been the key metabolite including a reduction into a double bond in the same ring as the lactone. This metabolite is also anticipated to be the key metabolite in organisms and its basis being a simple oxidation, and reduction, respectively. This metabolite may be excreted as such via the kidneys based on its log Kow of 3. It is more likely that this lactone will be reduced into an alcohol or de-esterified resulting in an acid and an alcohol and subsequently conjugation in the Phase 2 metabolic pathway. Via this pathway the kidneys will be the key excretion route as is seen in the repeated dose toxicity studies. Air is therefore not the key excretion route (e.g in contrast to fish) and therefore there is no concern for air-breathing organisms. This is explained in more detail by Gobas et al. (2020). They present that Oxygen containing chemicals are unlikely a concern for air-breathing organisms, because of their metabolism and kidneys being the key excretion pathways (see Toxico-kinetic section for more details).

Additional information

BCF study of Cassiffix (OECD TG 305)

The bioaccumulation (BCF) of the substance in Cyprinus carpio (common carp) was investigated according to OECD TG 305 in a flow-through test in compliance with GLP. Method: Stock solutions were prepared using N,N-Dimethylformamide. A solvent control group and single test concentration group of 20.0 µg/L (Constituent 1: 6.478 µg/L, Constituent 2: 3.540 µg/L, Constituent 3: 2.088 µg/L and Constituent 4: 1.494 µg/L) were used for 28 days as part of the uptake phase. Following confirmation of steady state, fish were then transferred to only test substance-free water during the depuration phase at day 29 till day 35.

Results: Average measured concentration in water were 5.77, 3.1, 1.87 and 1.28 ug/l for constituents 1-4, respectively. The test item concentrations were maintained ≥ 80% of the nominal test concentration and the variations were within ±20% of the average measured concentrations. Measured concentration in fish after 28 days of uptake were 1351, 787, 489 and 487 ug/kg, respectively. Steady-state concentrations of the substance were achieved in the tissues of common carp after 18 days of uptake. BCFss at day 28 were 230, 250, 260 and 380 for constituents 1-4, respectively. At day 29 day fish were transferred into clean water. Measured concentration during depuration in fish starting measuring at day 32 and 3 days thereafter. After day 35 the test concentrations in fish were 20, 15, 8.7 and 44.7 ug/kg fish, for constituent 1-4, respectively. Rapid depuration was observed, with a depuration half-life (t_1/2) of 0.8 days and depuration of 95% (t_0.95) ranged between 3.3 -3.6 days. Depuration parameter, k2, was calculated with the sequential method because first-order kinetics could be assumed (since no indications for deviations were observed; that is a linear relationship Ln concentration decrease over time). The k1/k2 values are: 231/0.899, 243/0.916, 230/0.838 and 351/0.842 and the BCFk are therefore 260, 270, 270 and 420 for constituent 1-4, respectively. The lipid content in fish were 3.92, 3.12 and 3.0% before uptake, after uptake and after depuration phase, respectively. For the lipid correction of the BCFss 3.12% is used resulting in a BCFssl of constituent 4 of 670 l/kg being the most conservative of all constituents. The average lipid content was 3.36%, which was used for the BCFk resulting in a BCFkl of 620 for constituent 4 being the most conservative value from the kinetic assessment. The BCF_KL for all four constituents derived with first order kinetics matched with their BCF_SSL.

Discussion: Lipid correction is applied for the derivation of the final BCF values. Growth correction was not applied for the derivation of the BCFk. Gobas & Lee (2019*) demonstrated the necessity to maintain mass balance in the derivation of the BCFk. Applying growth correction only for the depuration phase would be a violation of mass balance during the full test. In addition, they demonstrated errors when deriving BCFk corrected for growth, when violating the mass balance systematics. Because BCFk values in the current study are relatively low (max 620 L/kg), errors when correcting for growth in the depuration phase would have been small. Also, depuration rates in the current study are much faster than growth rate, resulting in a minor difference in the kinetic BCF even when correction for growth would have been applied. In addition, the BCFssl for the constituents are slightly more conservative compared to their BCFkl. The BCFssl of constituent 4 is slightly more conservative compared to the constituent 1, 2 and 3. Therefore the BCF of constituent 4 is used for the overall conclusion. The BCF_SSL for constituent 4 is also slightly more conservative compared to BCFkl of constituent 4: 670 versus 620 l/kg. The overall BCF_SSL of 670 l/kg will therefore be used for the risk assessment.

*Gobas, F.A.P.C. and Y-S. Lee, 2019. Growth‐Correcting the Bioconcentration Factor and Biomagnification Factor in Bioaccumulation Assessments. Environmental Toxicology and Chemistry—Volume 38, Number 9—pp. 2065–2072.