Pentyl salicylate

Bioaccumulation: aquatic / sediment

Description of key information

The bioaccumulation potential of amyl salicylate has been assessed using a read-across approach supported by in vitro metabolism data for both amyl salicylate and the analogue substance for which a BCF value is available, cyclohexyl salicylate. In applying the read-across two important aspects have been considered: the similar potential for metabolism of the two substances and the slight difference in lipophilicity (as modelled by logKow). As suggested in ECHA guidance R.7.10.3.2, the BCF value for the analogue has been corrected by the same factor of difference as for Kow to give a reasonable realistic estimate for amyl salicylate of 380-570 L/kg.

Key value for chemical safety assessment

BCF (aquatic species):

570 L/kg ww

Additional information

Reliable measured aquatic bioaccumulation data is not available for amyl salicylate. 

According to REACH Annex IX, information on bioaccumulation in aquatic species, preferably fish, is required for substances manufactured or imported in quantities of 100 t/y or more unless the substance has a low potential for bioaccumulation (for instance a log Kow ≤ 3). However,REACH Annex XI encourages the use of alternative information before a new vertebrate test, including fish, is conducted.

Amyl salicylate has a measured log Kow of >3. A read-across approach has been used to assess the bioaccumulation potential of amyl salicylate since a valid BCF value of 600-900 L/kg is available for an analogue substance, cyclohexyl salicylate. This is an analogue approach for which the read-across hypothesis is that the target substance (amyl salicylate) and source substance (cyclohexyl salicylate) have similar aquatic bioaccumulation as a result of structural similarity, expected similar metabolic potential in fish and limited differences in lipophilicity (as modelled by log Kow).

The two substances are structurally closely related as both are alkyl 2-hydroxybenzoate esters. The only slight structural difference is the nature of the alkyl group (5 versus 6 carbon atoms; linear versus cyclic). The target substance is a mixture of two isomers, which are expected to have similar aquatic bioaccumulation potential. The source substance is a mono-constituent. They do not contain any impurities that are expected to affect their bioaccumulation properties.

In applying the read-across two important aspects have been considered, i.e. the lipophilicity and the metabolic activity of both substances.

The BCF value of a substance is generally positively correlated with its lipophilicity (as modelled by log Kow). ECHA guidance R.7.10.3.2 advocates the application of a correction factor as long as the difference in log Kow is limited and gives the following example: “if the substance to be evaluated has one methyl group more than the compound for which a BCF value is available, the log Kow will be 0.5 higher and the estimated BCF from read-across is derived from the known BCF multiplied by a factor of 10^0.5. Amyl salicylate has a measured log Kow value of 4.4 to 4.5. Cyclohexyl salicylate has a log Kow value that is 0.2 log units higher. Therefore, an adjustment factor of 10^0.2(i.e. 1.58) has been applied to the cyclohexyl salicylate measured BCF value of 600-900 L/kg to give a reasonable realistic estimate for amyl salicylate of 380-570 L/kg.

The capability of fish to metabolise substances to more polar components, leads to lower BCF values. When applying read-across it is important to examine the potential for metabolism for the substance to be evaluated and the analogue substance for which the BCF value is available. In fish as in mammals, the liver is the principle organ of chemical biotransformation. It is reasonable, therefore, to evaluate whole animal metabolism using in vitro systems derived from liver tissue.

An in vitro metabolism assay using trout liver S9 fractions has been conducted on amyl salicylate and cyclohexyl salicylate. Metabolic stability was determined by monitoring the disappearance of the test item as a function of time. Incubations conducted using heat-denatured S9 were used to distinguish between enzymatic metabolism and other potential loss processes such as abiotic degradation, volatilization and adsorption to the reaction vessel. Amyl salicylate gives a chromatographic profile consisting of two major peaks, which were quantified separately (peak 1 = 28% abundance, peak 2 = 72% abundance at time 0 with the GC-method used). Cyclohexyl salicylate gives a chromatographic profile consisting of one peak.

Rapid turnover of both substances was observed over 20 minutes. Peak 1 and peak 2 of amyl salicylate demonstrated metabolic turnover rates respectively of 63% and 81% of the starting concentration within a 20 minute exposure period. Cyclohexyl salicylate demonstrated an average metabolic turnover of 69% over the same time period. The in vitro intrinsic clearance rate (CL_{int, in vitro}) was calculated from the log-transformed measured concentrations of parent compound as a function of time in two independent experiments. The average reaction rate was 3.2 ml/h/mg protein for peak 1 of amyl salicylate, 6.2 ml/h/mg protein for peak 2 of amyl salicylate and 3.5 ml/h/mg protein for cyclohexyl salicylate. In summary, peak 1 of amyl salicylate exhibited similar in vitro metabolic activity to cyclohexyl salicylate, while peak 2 of amyl salicylate was more rapidly metabolized. The increased in vitro metabolism observed for peak 2 of amyl salicylate (major constituent, n-pentyl salicylate) compared to the peak 1 of amyl salicylate (minor constituent, 2-methylbutyl salicylate) and cyclohexyl salicylate might be attributed to the small structural differences in the alkyl group (i.e. straight-chain versus branched-chain and cyclic). For example, the branched and cyclic alkyl groups may result in some inhibition of metabolism at the centre of metabolic action.

The in vitro metabolism data for amyl salicylate and cyclohexyl salicylate was used in the “in vitro - in vivo” extrapolation model developed by Nichols to refine the estimation of a partitioning based BCF. A binding term, fu, is used to correct for the difference in free chemical concentration between blood and the in vitro system. Two assumptions are possible: 1) fu can be calculated as the ratio of predicted free fractions in plasma and in the in vitro system using log Kow based algorithms, or 2) binding in vitro and in vivo can be assumed to be equal ( fu = 1.0). The refined BCF was estimated using both approaches.For cyclohexyl salicylate the estimated values were 329 L/kg (fu calculated) and 144 L/kg (fu = 1). The refined BCF estimates for amyl salicylate were slightly lower at 254 (peak 1, fu calculated), 209 L/kg (peak 2, fu calculated), 116 (peak 1, fu = 1) and 123 (peak 2, fu = 1). Although comparison with the in vivo data for cyclohexyl salicylate (BCF = 600-900 L/kg) indicates that the “in vitro – in vivo” extrapolation model underestimates the BCF value by a factor of approximately 2 to 6 for this class of chemical, the relative refined BCF estimates for amyl salicylate and cyclohexyl salicylate, which take into consideration both the log Kow and in vitro metabolic clearance of the substance, do support a lower in vivo BCF value for amyl salicylate compared to cyclohexyl salicylate.

Further indicators for similar potential for metabolism between the target and source substance include:

-         Both substances are readily biodegradable and thus are likely to be rapidly metabolised in organisms.

-         The two substances share common “bioaccumulation metabolism alert substructures” as profiled in the OECD QSAR Toolbox version 3.0 and the BCFBAF v3.1 whole body primary biotransformation rate constant model for fish. The main potential sites of metabolic attack, indicated by the negative coefficient values, are expected to be the same.

Indicators of the bioaccumulation potential for amyl salicylate and cyclohexyl salicylate are summarised in the table below:   

Indicator of bioaccumulation potential		Amyl Salicylate		Cyclohexyl Salicylate
Ready Biodegradability		Readily		Readily
Bioaccumulation – metabolism alerts (OECD QSAR toolbox v 3.0; BCFBAF v3.01).		Number of each fragment in substance
Fragment	Coefficient^(1)	Peak 1 (minor)	Peak 2 (major)
Ester [-C(=O)-O-C]	-0.7605	1	1	1
Aromatic alcohol [OH]	-0.4727	1	1	1
Benzene	-0.4277	1	1	1
-CH- [linear]	-0.1912	1	0	0
-CH- [cyclic]	0.0126	0	0	1
-CH2- [linear]	0.0242	2	4	0
Linear C4 terminal [CCC-CH3]	0.0341	0	1	0
-CH2- [cyclic]	0.0963	0	0	5
Methyl [-CH3]	0.2451	2	1	0
Aromatic H	0.2664	4	4	4
Partition coefficient N-Octanol/water (Log Kow)^(2)		4.4	4.5	4.7
In vitro S9 results^(3)
Metabolic turnover (% decrease, 20 minutes incubation)		63	81	69
In vitro intrinsic clearance rate (ml/h/mg protein)		3.2	6.2	3.5
In vitro – in vivo extrapolation model (Nichols et al)
Refined BCF (fu=1), L/kg		116	123	144
Refined BCF (fu calculated), L/kg		254	209	329
Measured BCF, L/kg				600-900

1) Descriptors with positive signs reflect a general reduced capacity for biotransformation and negative values reflect a general increased capacity for biotransformation; 2) Determined according to OECD 107 guideline. Identical conditions were used for the target and source substance (i.e. HPLC column, mobile phase, reference substances) ; 3) Average, based on two independent experiments.

Based on the above information, it is concluded that there is no need for further investigation of aquatic bioaccumulation with fish. 

For classification purposes, an experimentally derived high quality log Kow value is suitable when a measured BCF on an aquatic organism is not available. Amyl salicylate has a measured log Kow of 4.4 to 4.5. This exceeds the CLP cut-off value of ≥ 4 and the DSD value of ≥ 3. Amyl salicylate has an estimated BCF value of 380-570 L/kg, based on read-across from analogue data and corrected for slight differences in log Kow. This is close to the CLP threshold value of ≥ 500 L/kg and exceeds the DSD criteria of ≥ 100 L/kg. Thus amyl salicylate is considered to have the potential to bioconcentrate for classification purposes.

For the PBT and vPvB assessment a screening criterion has been established, which is log Kow greater than 4.5. Amyl salicylate has a measured log Kow of 4.4 to 4.5, which is just below this screening criterion. For organic substances with a log Kow below 4.5 it is assumed that the affinity for the lipids of an organism is insufficient to exceed the B criterion, i.e. a BCF value of 2000 L/kg. Since there is evidence that amyl salicylate is metabolised in fish, the aquatic BCF of amyl salicylate is expected to be lower than that predicted from log Kow. Indeed, the estimated BCF value for Amyl Salicyate based on read-across from analogue data is in the range of 380 to 570 L/kg. This is well below the B definitive criterion of BCF > 2000 L/kg. Therefore amyl salicylate does not fulfil the B-criteria.

The estimated BCF value for Amyl Salicyate based on read-across from analogue data is in the range of 380 to 570 L/kg. The highest value of 570 L/kg has been chosen as a relevant and reliable conservative estimate for risk assessment purposes.