2-tert-pentylcyclohexyl acetate

Bioaccumulation: aquatic / sediment

Description of key information

The bioaccumulation potential is assessed based on read-across from the close structural analogue Verdox (CAS# 20298-69-5) and the fish BCF value determined at 156 L/kg w.w. (normalised lipid fraction).

Key value for chemical safety assessment

BCF (aquatic species):

156 L/kg ww

Additional information

In this section the BCF study of the analogue substance Verdox will be presented and thereafter the justification for this read across. The section however will start with an abstract of a poster in which the metabolism of Coniferan in S9 and in cryopreserved hepatocytes is tested. This in vitro information is considered supporting, however, because it is information on Coniferan, this section will start with it.

Coniferan_In vitro metabolism:

The rainbow trout S9 and cryohepatocyte metabolic systems have been characterized elsewhere (Johanning et al., SETAC 2008 and SETAC 2009) demonstrating the activity of several important Phase I and Phase II enzymes. The test chemical Coniferan was incubated with male rainbow trout (1+) cryohepatocytes or S9 fraction and cofactors (0.1 mM PAPS, 2 μM UDPGA and 1 mM NADPH) at pH =7.8. In a water bath at 12°C Hepatocyte incubations were performed with both radioactive and non-radioactive Coniferan. All incubations were performed in triplicate at two or three different test chemical concentrations, and with hepatocytes at 0.5 X 106 cells/mL or S9 protein at 0.2 and 1 mg/L concentrations. All incubations included six time points. Coniferan reactions were terminated by the addition of ethyl acetate. Incubation samples were vortexed, centrifuged at 3000 rpm for 10 min at room temperature. The organic layer was transferred to a GC test vial and test chemical disappearance was measured by GC/MS. In all cases, zero-time incubations, heat-treated (inactivated, boiled at 100°C for 10 min), solvent control (SC) and no cofactors (NC) (all controls incubated for the longest time point) samples were included. Results were reported as

disappearance of test chemicals over time. Metabolites were examined by GC/MS and HPLC-β-RAM. Chromatograms were compared to those from tissue extracts of rainbow trout exposed to the same chemicals. Tissue homogenates from fish fed radiolabeled Coniferan (data not shown) over 28 days were analyzed. Homogenates were extracted with two volumes of ethyl acetate and samples of the organic phase after spinning at 3000 rpm for 5 minutes, were examined by GC-MS.

Radiolabeled and non-labeled Coniferan was incubated in the presence of rainbow trout liver S9 fractions and cryopreserved hepatocytes. Very little difference in the extent of the metabolic turnover of each of the four compounds was found between the liver S9 fraction and hepatocyte systems. The liver S9 incubations of Coniferan showed three metabolites with two of these found in the hepatocyte incubations. Two main metabolites from Coniferan appear to be deacetylation products.

Analogue Verdox_BCF information

The BCF of the analogue Verdox in rainbow trout (Oncorhynchus mykiss) was determined in a GLP-compliant OECD guideline 305 study. In this flow-through test, groups of 85 fish were exposed for a 33 day uptake phase to nominal test concentrations of 0, 1.7 and 17 µg/L of the test substance followed by a 10 day depuration phase. Steady-state concentrations of 14C-labeled Verdox were achieved in the tissues of rainbow trout (Oncorhynchus mykiss) after 33 days. The mean measured water concentrations based on total radioactivity were 2.3 and 29 μg/L. Steady-state BCF values for the 2.3 µg/L test concentration, based on total radioactivity Verdox concentrations were 65, 335 and 179 in edible, non-edible and whole fish tissue, respectively. Steady-state BCF values for the 29 µg/L test concentration, based on total radioactivity Verdox concentrations were 66, 402 and 203 in edible, non-edible and whole fish tissue, respectively. Verdox depurated quickly in fish tissue and ranged from 0.5 to 7% of Day 33 steady-state values by Day 10 of depuration. Kinetic BCFK values derived by nonlinear regression for the 2.3 µg/L treatment group were 65, 312 and 167 for edible, non-edible and whole fish tissue, respectively. The time to reach 90% steady state based on kinetics was 13.5, 5.3 and 6.6 days and time to reach 50% clearance was 4.06, 1.61 and 2.00 days for edible, non-edible and whole fish tissue, respectively. The worst case BCF of 203 for whole fish can be converted to a standard fish lipid content of 5%. Thus a BCF of 203/6.5*5 = 156 is used for further evaluations.

Read across justification

Bioaccumulation of Coniferan (CAS #67874-72-0) using read across from data available from Verdox (CAS #20298-69-5).

Introduction and hypothesis for the analogue approach

Coniferanis an acetate-ester attached to a cyclohexyl ring with a tert-pentyl-group attached at the ortho-position. Coniferan has a log Kow of >5 and therefore potential for bioaccumulation is to be anticipated. Experimental information is available only from anin vitrohepatocyte study with Coniferan, but this data alone is considered insufficient for adequate assessment of the bioaccumulation potential. Further information is needed.

In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such asin vitrotests, QSARs, grouping and read-across. For the assessment of the bioaccumulation potential of Coniferan, the analogue approach is selected because for a closely related analogue, Verdox,in vivoexperimental bioaccumulation information is available which can be used for read across.

Hypothesis: Coniferan has similar bioaccumulation potential as Verdox as the twosubstances are very close structural analogues. There are no indications that bioaccumulation of both substances is triggered by mechanisms other than lipophilicity, such as, for example, active transport mechanisms. Therefore, the substances’ octanol-water partitioning coefficients (log Kow) are considered to give direct indication of their bioaccumulation potential.

Available information:ForConiferan, a log Kow of 5.4 is available andin vitrodata addressing BCF in relation to metabolisms as presented on a poster with sufficient detail to allocate it Klimisch 2 (Weeks et al., 2009). In this study80% Coniferan is metabolised after 120 minutes incubation indicating that the substance is readily metabolised in fish. DT50 is around 20 minutes and the detected metabolite is the alcohol of Coniferan (the ester is cleaved, see Fig. 1).

For theanalogue Verdox, a BCF value form a fish study according to OECD TG 305 is available. The data are reliable without restrictions (Klimisch 1). In this flow-through test, groups of 85 rainbow trout were exposed for a 33-day uptake phase to nominal test concentrations of 0, 1.7 and 17 µg/L of the test substance followed by a 10 day depuration phase. Steady-state concentrations of 14C-labelled Verdox were achieved in fish tissues after 33 days. The mean measured water concentrations, based on total radioactivity, were 2.3 and 29 μg/L. Steady-state BCF values for the 2.3 µg/L test concentration, based on total radioactivity Verdox concentrations, were 65, 335 and 179 in edible, non-edible and whole fish tissue, respectively. Steady-state BCF values for the 29 µg/L test concentration, based on total radioactivity Verdox concentrations, were 66, 402 and 203 L/kg in edible, non-edible and whole fish tissue, respectively. Verdox depurated quickly in fish tissue and ranged from 0.5 to 7% of Day 33 steady-state values by Day 10 of depuration. Kinetic BCFK values derived by nonlinear regression for the 2.3 µg/L treatment group were 65, 312 and 167 L/kg for edible, non-edible and whole fish tissue, respectively. The time to reach 90% steady state based on kinetics was 13.5, 5.3 and 6.6 days and time to reach 50% clearance was 4.06, 1.61 and 2.00 days for edible, non-edible and whole fish tissue, respectively. The worst case BCF of 203 L/kg for whole fish was converted to a standard fish lipid content of 5% (i.e. 203/6.5*5) to derive a BCF of 156 L/kg. This value was used for further evaluations.

Target chemical and source chemical

Chemical structures and physico-chemical properties of the target chemical and the source chemical are shown in the data matrix.

Purity / Impurities

Coniferan is a multi-constituent containing two stereo isomers. The purity of Coniferan is close to 100%. In view of Verdox also being a multi-constituent containing two stereo isomers and having a purity close to 100%, there will be no significant impurities relevant for read across.

Analogue approach justification

According to Annex XI section 1.5, read across can be used to replace testing when the similarity can be used on a common backbone and a common functional group. Verdox is selected as the analogue because it is the closest related analogue to be found.

In accordance with ECHA guidance (2017, RAAF) Verdox was selected as an analogue in with a difference of only one methyl group and for which BCF information was available.

Structural similarities and differences:Coniferan and Verdox show very close structural resemblance. Both are esters with acommon cyclohexyl acetate backbone.Coniferan has a one additional methyl group attached to the tert-butyl group. i.e. it contains a tert-pentyl group (see data matrix for chemical structures).

Bioavailability: Both substances are esters and present similar bioavailability based on their physico-chemical properties. The vapour pressure and water solubility of Coniferan are slightly lower and the log Kow is slightly higher than that of Verdox which may be attributable to the extra methyl group of Coniferan.

Metabolism: Coniferan and Verdox are both acetic esters and their acetic groups will be readily cleaved by carboxyl-esterases in fish under formation of the respective alcohols and acetic acid (see metabolic scheme below) (Wheelock et al. 2009). Based on thein vitrohepatocyte findings with Coniferan (Weeks et al., 2009) and the Verdoxin vivoBCF study both substances are readily metabolised. Therefore the same BCF is anticipated for both substances and no conversion factor, based on e.g. log Kow, is needed.

 

Fig. 1 . The anticipated metabolism of Coniferan (1st structure) and Verdox (2nd structure) into their anticipated alcohols and acetic acid.

Other experimental data that can be used for support:For another similar ester (Cyclabute: CAS#93941-73-2)anin vivoBCF of ca. 200 was determined further confirming the read-across considerations presented here (this information will not be further addressed in this read-across justification but will be presented in the Annex VIII registration of Cyclabute).

Uncertainty of the prediction:In view of the close similarity in backbone and functional group, bioavailability (based on physico-chemical properties) and metabolism, there no remaining uncertainties. In addition, metabolism is experimentally confirmedin vitroandin vivo, because the BCF solely based on log Kow are much higher. Also QSAR predictions that include metabolism present that a low BCF is expected (BCFBAF predicts 811 and 385, for Coniferan and Verdox respectively). The experimental BCF of Verdox, 156, is nicely aligned with the predicted BCF of its alcohol: 83 (based on its predicted log Kow of 3.4, which indicates that the key metabolite is the alcohol. Using the criteria of reliability in the ECHA guidance (2017, RAAF) the score 5 is applicable based on the reasoning above.

Data matrix

The relevant information on physico-chemical properties and other environmental fate properties are presented in the Data Matrix below.

Conclusions for hazard, environmental classification and labelling and risk characterisation

Hazard assessment: For the structural analogue Verdox a BCF of 156 is derived. In view of the similarities between Verdox and Coniferan in view of structure, bioavailability and metabolic features, the same BCF value for Coniferan will be used.

Classification and labelling: This BCF value does not influence the classification and labelling according toregulation EC/1272/2008 (CLP) because the substance has acute aquatic toxicity between 1 and 10 mg/l, a NOEC for algae of 0.5 mg/l and is‘not rapidly degradable’.

PBT assessment:For Coniferan the BCF of 156 L/kg, as determined from read-across, is well below the cut-off value of 2000 for ‘Bioaccumulative (B)’. The substance is therefore assessed as ‘not B/vB’.

Risk characterisation: The BCF value of 156 L/kg will be used in the environmental exposure assessment.

Data matrix for read across

Common names	Coniferan	Verdox
Chemical name	2-tert-pentylcyclohexyl acetate	2-tert-butylcyclohexyl acetate
Chemical structures
CAS No.	67874-72-0	20298-69-5
EC no	Registration 2018	243-718-1 (registered)
Empirical formula	C13H24O2	C12H22O2
Physico-chemical data
Molecular weight	212.3	198.3
Physical state	liquid	Liquid at 30°C
Melting point,^oC	-20	29.8
Boiling point,^oC	252	232
Vapour pressure, Pa	4.24 (at 24 °C)	9.72 (at 23 °C)
Water solubility, mg/L	7.6 (at 24 °C)	10 (at 23 °C)
Log Kow	5.4	4.8
Environmental fate data
Biodegradation	Not readily	Not readily
Hydrolysis	No data	DT50 = 42 - 49 hours
Adsorption-/desorption	No data	Log Koc = 3.12 (Koc = 1300 L/kg)
BCF	Read across from Verdox:	Experimental data: 156 L/kg (OECD TG 305)

References:

Wheelock, C.E., Philips, B.M., Anderson, B.S., Miller, J.L., Miller, M.J., and Hammock, B.D., 2008, Application of carboxylesterase activity in environmental monitoring and toxicity identification evaluations, (TIEs), in Reviews of Environmental Contamination an Toxicology, ed. Whitacre, 117-178, D.M., Springer.

Weeks, J.A., Guiney, S.C., Racine, W.I., Johannig, K.M., Hill, J.E., Gauvin, R.L., Holden, D., J.E., Hill, R, 2009, Vitro Fish Metabolism and Metabolite Profiling of Several Fragrances using Rainbow Trout Liver S9 Fraction and Cryopreserved Hepatocytes. Poster presentation at SETAC,

http://tools.thermofisher.com/content/sfs/brochures/setaceu2010invitrofishmetabolismandmetaboliteprofilingusingrainbowtroutlivers9fractionandcryopreservedheps.pdf)