Reaction mass of 2-methylbutyl salicylate and pentyl salicylate

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid BCF is available for the analogue substance, cyclohexyl salicylate (GLP study in accordance with EU Method C.13). As the study is being used in a read-across approach, it is rated reliability 2 (the maximum for read-across). The read-across is an analogue approach based on the hypothesis that amyl salicylate (the registration substance) and cyclohexyl salicylate (source substance) have similar aquatic bioaccumulation potential as a result of structural similarity, expected similar metabolic potential in fish and limited differences in lipophilicity (as modelled by log Kow).

Qualifier:

according to guideline

Guideline:

EU Method C.13 (Bioconcentration: Flow-through fish test)

Version / remarks:

EEC-Directive 79/831, Annex V, Part C

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 0.055Pa at 20'C (Gas Saturation method, data submitter is data owner)
- Water solubility: 5.3mg/l at 20'C (OECD 105, data submitter is data owner)
- log Pow: 4.7 (OECD 117, data submitter is data owner)

OTHER PROPERTIES (relevant for this endpoint)
- Ready biodegradability: Readily biodegradable - 87% biodegradation after 28 days with the 10-day window fulfilled (OECD 301F, data submitter is data owner)

Radiolabelling:

yes

Details on sampling:

Fish were sampled from the two basins at regular intervals to measure the concentration of 14C-labelled test substance in the tissues.

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: automatic dosing system adding proportionate volumes of test substance solution and dilution water to the mixing vessel; the mixing ratio of test substance concentrate and dilution water is 1:100
- Controls: not required since 14C-labelled test material was used
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): 100-times concentrates contained 0.1 mL ethanol per one concentrate
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: West Aquarium GmbH
- Age at study initiation (mean and range, SD): not given
- Length at study initiation (lenght definition, mean, range and SD): about 3 cm
- Weight at study initiation (mean and range, SD): 0.219 ± 0.05 g for fish used for lipid content determination
- Weight at termination (mean and range, SD): 0.295 ± 0.07 g for fish used for lipid content determination
- Health status: low mortality of < 1% during the last two weeks before study start
- Description of housing/holding area: test basins with 6 L volume (minimal volume 3 L), about 10 water changes per day, automatic cleaning and removal of food residues and faeces, constant temperature of 23 °C ± 1 °C

FEEDING DURING STUDY
- Food type: Altromin fish food N-1324
- Amount: about 2% of actual fish wet weight
- Frequency: automatically

ACCLIMATION
- Acclimation period: fish were delivered about one month before study start, 2 days before start the fish were placed in the test basins

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

not reported

Test temperature:

Basin 1: 23 °C ± 0.13 °C
Basin 2: 23.1 °C ± 0.12 °C

pH:

Basin 1: 7.51 ± 0.07
Basin 2: 7.50 ± 0.08

Dissolved oxygen:

Basin 1: 79.9% of saturation ± 8.3%
Basin 2: 79.1% of saturation ± 9.3%

TOC:

not reported

Salinity:

not applicable

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: glass vessels, 6 L fill volume
- Aeration: continuous aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 10 renewals per day
- No. of organisms per vessel: Basin 1 contained 55 and Basin 2 40 fish
- No. of vessels per concentration (replicates): 1
- Biomass loading rate: 3 g fish wet weight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water filtered through active carbon
- Composition of test medium: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: not reported

Nominal and measured concentrations:

Nominal concentrations: 1 and 10 µg/L (Basin 1 and Basin 2, respectively)
Mean measured concentrations (measured with 14C activity determination): 1.05 and 8.5 µg/L

Reference substance (positive control):

no

Lipid content:

13.92 %

Time point:

start of exposure

Lipid content:

9.35 %

Time point:

end of exposure

Type:

BCF

Value:

1 136

Basis:

whole body w.w.

Time of plateau:

5 d

Remarks on result:

other: total radioactivity, read-across substance

Remarks:

Conc.in environment / dose:1 µg/L

Type:

BCF

Value:

1 170

Basis:

whole body w.w.

Time of plateau:

5 d

Remarks on result:

other: total radioactivity, read-across substance

Remarks:

Conc.in environment / dose:10 µg/L

Type:

BCF

Value:

600 - 900

Basis:

other: TLC analysis of ethanol extracts of fish showed the greatest portion of the extracted radioactivity (ca. 50 to 75%) was the unchanged test substance. From the analytical determinations, a BCF of 600 to 900 was calculated for the parent substance.

Remarks on result:

other: parent specific analysis, read-across substance

Type:

BCF

Value:

380 - 570

Remarks on result:

other: Estimated, amyl salicylate. Adjustment factor of 1.58 applied to the read-across substance values to take into account differences in log Kow.

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

2.5 d

Details on kinetic parameters:

Not calculated, but the depuration half-life was clearly below 3 days in the study

Metabolites:

Not investigated

Results with reference substance (positive control):

Not applicable

Details on results:

ANALOGUE APPROACH JUSTIFICATION: See attached "Aquatic Bioaccumulation Read-Across Justification for Amyl Salicylate" document for full details. In summary, the read-across is an analogue approach based on the hypothesis that the target substance (amyl salicylate) and source substance (cyclohexyl salicylate) have similar aquatic bioaccumulation potential as a result of structural similarity, expected similar metabolic potential in fish and limited differences in lipophilicity (as modelled by log Kow). Important considerations for the use of the read-across are:
- The target substance is a mixture of two isomers, which are expected to have similar aquatic bioaccumulation potential. The source substance is a monoconstituent. The target and source substance have typical purities of => 98.5% (sum of isomers) and >=99.8% respectively. They do not contain any impurities that are expected to affect their bioaccumulation properties.
- The target and source substance are structurally closely related as both are alkyl 2-hydroxybenzoate esters. The only structural difference is the nature of the alkyl group (5 versus 6 carbon atoms; linear/branched versus cyclic), which is not expected to have a significant effect on the aquatic bioaccumulation potential of the two substances.
- In applying the read-across two important aspects have been considered, i.e. the metabolic activity and the lipophilicity of both substances.
METABOLIC ACTIVITY: Both substances are expected to have similar bioaccumulation potential in fish which is supported by the following indicators, i-iii (see also table 1). There is some indication (based on in vitro data for the major isomer, point ii below, and predicted kM for both isomers, point iii below) that amyl Salicylate (target substance) may have a slightly higher potential for metabolism than cyclohexyl salicylate (source substance) which indicates that the read-across may be conservative.
(i) Both substances are readily biodegradable and thus are likely to be rapidly metabolised in organisms.
(ii) An in vitro metabolism assay using trout liver S9 fractions has been conducted on both substances (see supporting studies for details). The in vitro intrinsic clearance rate (CLint, in vitro) was determined to be 3.2 ml/h/mg protein for peak 1 of amyl salicylate (corresponding to the 2-methyl butyl salicylate isomer), 6.2 ml/h/mg protein for peak 2 of amyl salicylate (pentyl salicylate isomer) and 3.5 ml/h/mg protein for cyclohexyl salicylate. The results show that the in vitro metabolic activity of cyclohexyl salicylate and one isomer of amyl salicylate are identical, while the second isomer of amyl salicylate is slightly more rapidly metabolised.
(iii) Both substances share common “bioaccumulation metabolism alert substructures” as profiled in the BCFBAF v3.1 whole body primary biotransformation rate constant (Km) model for fish. Both substances contain an “ester”, “aromatic alcohol” and "benzene" structural fragment, which are considered potential sites of metabolic attack as indicated by the negative coefficient values. The predicted Km values indicate that the two isomers of amyl salicylate (kM=6.42 minor isomer; kM=5.60 major isomer) may have a slightly higher potential for metabolism than cyclohexyl salicylate (kM=3.98/day). This is a result of the different fragments associated with the alkyl substituents (i.e. cyclohexyl versus amyl) used by the model and higher log Kow of cyclohexyl salicylate.
LIPOPHILICITY: There is a limited difference in the log Kow values of the two substances. Since amyl salicylate has a lower measured logKow value than the source substance (4.4-4.5 compared to 4.7 ) direct read-across represents a worst-case scenario (i.e. amyl salicylate is likely to have a lower in vivo BCF value).

CORRECTED BCF VALUE:
- 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 to the power of 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 to the power of 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 worst-case estimate for benzyl salicylate of 380-570 L/kg.

Table 1: Summary of indicators of the bioaccumulation potential for amyl salicylate (target substance) and cyclohexyl salicylate (source substance) that support the read-across approach.

  

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
Whole body primary biotransformation rate constant (kM) for 10g fish, /day^(2)		6.42	5.60	3.98
Partition coefficient N-Octanol/water (Log Kow)^(3)		4.4	4.5	4.7
In vitro S9 intrinsic clearance rate (ml/h/mg protein)		3.2	6.2	3.5

 

1) Descriptors with positive signs reflect a general reduced capacity for biotransformation and negative values reflect a general increased capacity for biotransformation

2) Model available in the BCFBAF v3.01 programme.

3) Determined according to OECD 107 guideline. Identical conditions were used for the target and source substance (i.e. HPLC column, mobile phase, reference substances)

Validity criteria fulfilled:

yes

Remarks:

The lipid content of fish at the end of the test was slightly lower than generally acceptable

Conclusions:

The bioconcentration factor for Cyclohexyl salicylate (analogue substance) was determined by using 14C-labelled test material. The experimentally determined values at test concentrations of 1 and 10 µg/L were 1136 and 1170, respectively based on total radioactivity. Analytical determination of the test substance concentrations in ethanol extracts of fish tissue by thin layer chromatography gave BCF values ranging from 600 to 900.
Amyl salicylate is less lipophilic than cyclohexyl salicylate. The difference in log Kow is 0.2 log units. Therefore, in line with ECHA guidance R.7.10.3.2 an adjustment factor of 10 to the power of 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 highest value of 570 L/kg has been chosen for the purpose of classification and labelling and/or risk assessment.

Executive summary:

The bioaccumulation potential of the radiolabelled test substance Cyclohexyl salicylate [Carboxyl 14-C] was determined under GLP in accordance with EU Method C.13 under flow-through conditions. Zebra fish (Danio rerio) of about 3 cm length and 0.2 to 0.3 g weight were used in the experiment. The lipid content of fish was about 14% at the begin of the study and decreased to 9.4% at the end of the study. This decrease was slightly above the generally acceptable decrease of 25%. Two test concentrations of nominal 1 and 10 µg/L were used in the accumulation phase over a period of 28 days. The measured time-weighted average concentrations were 1.05 and 8.5 µg/L, respectively. Fish were sampled at regular intervals, put into liquid nitrogen, weighed and measured. The animals were burned in an oxidiser and the concentration of radiolabelled substance was measured with a liquid scintillation counter. The plateau was reached quickly after a few days (less than 5). The experimentally determined BCF values obtained with the test concentrations of 1 and 10 µg/L were 1136 and 1170, respectively.

After 28 days, a 14-day depuration phase was started by placing the test fish in basins with dilution water not containing the test substance. A rapid depuration was seen and the corresponding depuration half-life was clearly less than 3 days. Analytical determination of the test substance concentrations in ethanol extracts of fish tissue by thin layer chromatography gave BCF values ranging from 600 to 900.

Overall, it can be concluded that the substance Cyclohexyl salicylate tends to rapidly bioconcentrate in the zebra fish and that a plateau is reached quickly within less than 5 days. However, also the depuration is very rapid and the depuration half-life is clearly less than 3 days. The experimentally determined bioconcentration factors are clearly below the threshold limit value of 2000 indicating bioaccumulation potential. The substance therefore should be considered as not bioaccumulative.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

other: Experimental in vitro results and estimated BCF by calculation

Adequacy of study:

supporting study

Study period:

12 June to 10 July 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The in vitro data, generated from a pre-validated method, are considered reliable and have been used to produce an estimated BCF using a scientifically valid extrapolation model.

Principles of method if other than guideline:

The bioaccumulation potential is estimated from an in vitro fish liver S9 standardised assay, pre-validated by a consortium under the coordination of HESI/ILSI (Johanning et al, 2012). In vitro metabolic stability is determined by monitoring the disappearance of the test item as a function of time. The rate of substance depletion is used as input into an "in vitro - in vivo" extrapolation model (Nichols et al, 2006) to generate an estimated BCF value for a "standardised fish" (one that weighs 10g, has a 5% lipid content, and is living at 15'C).

GLP compliance:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were purchased from Lifetechnologies (formerly CellzDirect/Invitrogen) (“Pooled Male Hatchery Rainbow Trout Liver S9”; product code CZDTRS9PL, Lot# TR015) and stored at -80°C. The average body weight of the fish used for the preparation of S9 fractions was 800 g.

Details on test conditions:

Initially, a range finding experiment was performed to determine the optimal incubation times to be used in the main experiments.

A stock solution of Amyl Salicylate (10 mM) was prepared freshly in methanol and diluted in water resulting in 10 μM solutions. Stock solutions of cofactors were prepared freshly in 0.1 M potassium phosphate buffer, pH 7.8. Alamethicin was dissolved in methanol (5 mg/ml; aliquots stored at -80°C) and diluted in buffer (250 μg/ml).

Rainbow Trout liver S9 fractions were thawed on ice. All incubations were performed in potassium phosphate buffer at pH 7.8 (0.1 M) in Hirschmann glass tubes in triplicate incubated at 12°C in a Thermomixer (Eppendorf) at 700 rpm. Active S9 fractions protein or heat inactivated protein as control (1 mg/ml) was preincubated on ice with alamethicin (final concentration: 25 μg/ml). Alamethicin is a pore-forming peptide antibiotic which permeabilises microsomal membranes and activates glucuronidation by allowing free transfer of UDPGA and glucuronide product across the membrane. After addition of cofactors for Phase I (NADPH, Nicotinamide adenine dinucleotide 2′-phosphate reduced) and Phase II enzymes (UDPGA, Uridine 5′-diphosphoglucuronic acid; PAPS, Adenosine 3′-phosphate 5′-phosphosulfate; GSH, reduced L-glutathione), the reaction was initiated by addition of the test substance. Final concentrations of cofactors, protein and test substance are listed in the table in the section entitled "any other information on materials and methods incl. tables".

In the range finding experiment, Amyl Salicylate (1 μM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 120 minutes. As controls, the test substance was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors or with active S9 protein in absence of any cofactors. Reactions were stopped at 0, 30 minutes and 120 minutes incubation by addition of acetonitrile (200 μl) containing Cyclohexyl Salicylate (1 μM) as internal standard to the Hirschmann tubes. Samples were extracted with MTBE (200 μl) in the same tubes by vortexing for 30 seconds, centrifuged to allow a better phase separation and separation of protein (Eppendorf centrifuge, 12 000 rpm, 5 min, room temperature) and subjected to GC-MS analysis. The two major peaks of Amyl Salicylate were quantified separately.

In the two independent main experiments (1st and 2nd main experiment), Amyl Salicylate (1 μM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 20 minutes as described above. Reactions were stopped at time 0, 2.5, 5, 10 minutes and 20 minutes. As control, the test substance was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors for 0 minutes and 20 minutes and in presence of active S9 protein in absence of any cofactors for 20 minutes. Reactions were stopped and extracted as described above.

Details on estimation of bioconcentration:

The model version used was “S9spreadsheet_4202012_standardfish_consensusver-1.xlsx”. The S9 in vitro substrate depletion rate is converted to a whole-fish metabolism rate constant (Kmetab) using a number of extrapolation and scaling factors. The estimated Kmetab value is then used to refine the partitioning based BCF model prediction. A binding term, fu, is used to correct for the difference in free chemical concentration between in vivo 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 logKow-based algorithms, or 2) binding in vitro and in vivo can be assumed to be equal ( fu = 1.0). The refined BCF has been estimated using both approaches for the 2 peaks of Amyl Salicylate.

Type:

BCF

Value:

254 L/kg

Remarks on result:

other: Refined BCF for isomer 1, including biotransformation rate estimates (fu calculated)

Type:

BCF

Value:

209 L/kg

Remarks on result:

other: Refined BCF for isomer 2, including biotransformation rate estimates (fu calculated)

Type:

BCF

Value:

116 L/kg

Remarks on result:

other: Refined BCF for isomer 1, including biotransformation rate estimates (fu =1)

Type:

BCF

Value:

123 L/kg

Remarks on result:

other: Refined BCF for isomer 2, including biotransformation rate estimates (fu =1)

Details on results:

A rapid decrease of Amyl Salicylate within 30 minutes (76.6% decrease for isomer 1 and 84.1% decrease for isomer 2) and an almost complete decrease within 120 minutes (90.5% and 85.8% decrease for isomer 1 and 2, respectively) was observed in the range finding experiment with active S9 protein. In the absence of any cofactors added, a slower decrease of Amyl Salicylate was observed (13.1% decrease for isomer 1 and 19.7% decrease for isomer 2 within 120 minutes). A slight decrease of the two isomers of Amyl Salicylate was observed in the control samples with heat inactivated protein probably due to evaporation and / or adsorption (11.4 and 8.1% decrease for isomer 1 and 2, respectively within 120 minutes) (Appendix 2).

Thus, incubations were carried out up to 20 minutes in the two main experiments. Enzymatic turnover of Amyl Salicylate was rapid in presence of cofactors. 62.5% and 63.3% decrease was observed for isomer 1 of Amyl Salicylate (2-methylbutyl 2-hydroxybenzoate) in 20 minutes in two independent experiments Figure 1a). Isomer 2 (pentyl 2-hydroxybenzoate) was more rapidly metabolized than isomer 2 with 82.6% and 79.9% decrease in 20 minutes (Figure 1b). A slow turnover of both Amyl Salicylate isomers was found with active S9 protein in the absence of cofactors (4.4% to 10.9% decrease in 20 minutes). A similar, slow decrease of the two Amyl Salicylate isomers was observed with inactive S9 protein (5.7% to 11.8% decrease in 20 minutes).

The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transformed measured concentrations of parent compound as a function of time in two independent experiments for the two isomers: 2.90 and 3.56 ml/h/mg protein for isomer 1 (2-methylbutyl 2-hydroxybenzoate) and 6.22 and 6.25 ml/h/mg protein for isomer 2 (pentyl 2-hydroxybenzoate) (Appendix 3).

The average in vitro substrate depletion rates (3.2 ml/h/mg protein for peak 1 of amyl salicylate, 6.2 ml/h/mg protein for peak 2) were used as input into the in vitro - in vivo extrapolation model to generate estimated BCF values (see Table 1 in the section "Any other information on results incl. tables").

Table 1: Refined BCF estimates calculated with the in vitro-in vivo extrapolation model [1]

	Amyl Salicylate Isomer 1 (2-methylbutyl 2-hydroxybenzoate)		Amyl Salicylate Isomer 2 (pentyl 2- hydroxybenzoate)
Parameter	fu calc [2]	fu=1.0 [3]	fu calc [2]	fu=1.0 [3]
Parameter: in vitro data
S9 concentration (CS9) (mg/mL)	1	1	1	1
Reaction rate (Rate) (1/h)	3.23	3.23	6.24	6.24
Input Parameter
LogKow	4.4	4.4	4.5	4.5
Calculated Parameters
Partitioning based BCF, assuming no metabolism (BCFp)	1256	1256	1581	1581
In vitrointrinsic clearance (CLIN VITRO,INT) (ml/h/mg protein)	4.0	4.0	7.7	7.7
In vivointrinsic clearance (CLIN VIVO,INT) (l/d/kg fish)	71.6	71.6	138.3	138.3
Scaled clearance for 10 g fish (CLIN VIVO,INT,10) (l/d/kg fish)	214	214	414	414
Corrected clearance for 10 g fish (CLIN VIVO,INT,10,CORR) (l/d/kg fish)	535	535	1034	1034
Hepatic clearance (CLH) (L/d/kg fish)	9.4	23.5	13.2	24.0
Whole-body metabolism rate (kMETAB) (1/d)	2.0	4.9	2.6	4.7
BCF,on a total conc. basis, w/out lipid norm.(BCFTOT) (l/kg)	254	116	209	123

¹J. Nichols, personal communication (version: “S9spreadsheet_4202012_standardfish_consensusver-1.xlsx”), based on a previous publication from Nichols et al (2006).

²fu, plasma binding correction term; “fu calc”, hepatic clearance is calculated taking into account a theoretically calculated difference between in vitro and in vivo binding

³fu, plasma binding correction term; “fu = 1.0”, hepatic clearance is calculated assuming equal in vitro and in vivo binding by setting fu = 1.0

Conclusions:

Metabolic turnover by trout liver S9 fractions was observed for the two isomers of Amyl Salicylate, indicating that Amyl Salicylate is expected to be metabolised in vivo. The capability of fish to metabolise substances to more polar components, leads to lower BCF values. Indeed, a partitioning based BCF estimate (assuming no metabolism) was reduced from 1256-1581 L/kg to 115-254 L/Kg by incorporating in vitro depletion rates into an "in vitro - in vivo" extrapolation model. These refined BCF estimates indicate that Amyl Salicylate is expected to have a low potential for bioaccumulation.

Executive summary:

Introduction: A study was performed to assess the in vitro stability of Amyl Salicylate in fish liver S9 fractions. The method followed was a standardised assay, prevalidated by a consortium under the coordination of HESI/ILSI.

 

Experimental: Following a preliminary range finding test, Amyl Salicylate (1 µM) was incubated in triplicate with trout liver S9 fraction (1 mg/ml) for 0, 2.5, 5, 10 and 20 minutes at 12°C in two main, independent experiments. Negative controls included incubation of the test substance with heat inactivated S9 protein.

The disappearance of Amyl Salicylate as a function of time was monitored using GC-MS analysis. The two isomers of Amyl Salicylate were quantified separately. The rate of substrate depletion was calculated within the linear range of parent decrease and used as input into an "in vitro - in vivo" extrapolation model to generate an estimated BCF for a "standardised fish".

 

Results: Isomer 1of Amyl Salicylate (2-methylbutyl 2-hydroxybenzoate, 28.4% abundance at time 0 with the GC-method used) demonstrated a metabolic turnover of 62.5 to 63.3% of the starting concentration within a 20 minute exposure period. Isomer 2 of Amyl Salicylate (pentyl 2-hydroxybenzoate, 71.7% abundance at time 0 with the GC-method used) was metabolized more rapidly with a metabolic turnover of 82.6 to 79.9% of the starting concentration within a 20 minute exposure period. In contrast, there was a slow decrease of both Amyl Salicylate isomers with the heat inactivated S9 control (5.7-11.8% decrease within a 20 minute exposure period.

The in vitro intrinsic clearance was calculated from the log-transform measured concentrations of the two isomers of the parent compound as a function of time in two independent experiments: 2.90 and 3.56ml/h/mg protein for isomer 1 (2-methylbutyl 2-hydroxybenzoate); 6.22 and 6.25ml/h/mg protein for isomer 2 (pentyl 2-hydroxybenzoate). The average in vitro intrinsic clearance rate (3.2 ml/h/mg protein for isomer 1, 6.2 ml/h/mg protein for isomer 2) was used as input into an in vitro-in vivo extrapolation model to generate a refined BCF estimate of 116 l/kg for isomer 1 and 123 l/kg for isomer 2 using an assumed fu = 1.0 (i.e. no effect of differential binding to serum) and 254 l/kg for isomer 1 and 209 l/kg for isomer 2 assuming different binding to serum in vivo versus in vitro (fu calc).

Conclusions:  Metabolic turnover by trout liver S9 fractions was observed for the two isomers of Amyl Salicylate, indicating that Amyl Salicylate is expected to be metabolised in vivo. The capability of fish to metabolise substances to more polar components, leads to lower BCF values. Indeed, a partitioning based BCF estimate (assuming no metabolism) was reduced from 1256-1581 L/kg to 115-254 L/Kg by incorporating in vitro depletion rates into an "in vitro - in vivo" extrapolation model. These refined BCF estimates indicate that the 2 isomers of Amyl Salicylate are expected to have a low potential for bioaccumulation.

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

other:

Adequacy of study:

supporting study

Study period:

12 April to 01 June 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The in vitro data, generated from a pre-validated method, are considered reliable and have been used to produce an estimated BCF using a scientifically valid extrapolation model.

Principles of method if other than guideline:

The bioaccumulation potential is estimated from an in vitro fish liver S9 standardised assay, pre-validated by a consortium under the coordination of HESI/ILSI (Johanning et al, 2012). In vitro metabolic stability is determined by monitoring the disappearance of the test item as a function of time. The rate of substance depletion may be used as input into an "in vitro - in vivo" extrapolation model to generate an estimated BCF value for a "standardised fish".

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 0.055Pa at 20'C (Gas Saturation method, data submitter is data owner)
- Water solubility: 5.3mg/l at 20'C (OECD 105, data submitter is data owner)
- log Pow: 4.7 (OECD 117, data submitter is data owner)

OTHER PROPERTIES (relevant for this endpoint)
- Ready biodegradability: Readily biodegradable - 87% biodegradation after 28 days with the 10-day window fulfilled (OECD 301F, data submitter is data owner)

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were purchased from Lifetechnologies (formerly CellzDirect/Invitrogen) (“Pooled Male Hatchery Rainbow Trout Liver S9”; product code CZDTRS9PL, Lot# TR015) and stored at -80°C. The average body weight of the fish used for the preparation of S9 fractions was 800 g.

Details on test conditions:

Initially, a range finding experiment was performed to determine the optimal incubation times to be used in the main experiments.

A stock solution of Cyclohexyl Salicylate (10 mM) was prepared freshly in methanol and diluted in water resulting in 10 μM solutions. Stock solutions of cofactors were prepared freshly in 0.1 M potassium phosphate buffer, pH 7.8. Alamethicin was dissolved in methanol (5 mg/ml; aliquots stored at -80°C) and diluted in buffer (250 μg/ml).

Rainbow Trout liver S9 fractions were thawed on ice. All incubations were performed in potassium phosphate buffer at pH 7.8 (0.1 M) in Hirschmann glass tubes in duplicate (range finding experiment) or triplicate (main experiemnts) incubated at 12°C in a Thermomixer (Eppendorf) at 700 rpm. Active S9 fractions protein or heat inactivated protein as control (1 mg/ml) was preincubated on ice with alamethicin (final concentration: 25 μg/ml). Alamethicin is a pore-forming peptide antibiotic which permeabilises microsomal membranes and activates glucuronidation by allowing free transfer of UDPGA and glucuronide product across the membrane. After addition of cofactors for Phase I (NADPH, Nicotinamide adenine dinucleotide 2′-phosphate reduced) and Phase II enzymes (UDPGA, Uridine 5′-diphosphoglucuronic acid; PAPS, Adenosine 3′-phosphate 5′-phosphosulfate; GSH, reduced L-glutathione), the reaction was initiated by addition of the test substance. Final concentrations of cofactors, protein and test substance are listed in the table in the section entitled "any other information on materials and methods incl. tables".

In the range finding experiment, Cyclohexyl Salicylate (1 μM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in duplicate for up to 120 minutes. As controls, the test substance was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors or with active S9 protein in absence of any cofactors. Reactions were stopped at 0, 30 minutes and 120 minutes incubation by addition of acetonitrile (200 μl) containing methyl laurate (1 μM) as internal standard to the Hirschmann tubes. Samples were extracted with MTBE (200 μl) in the same tubes by vortexing for 30 seconds, centrifuged to allow a better phase separation and separation of protein (Eppendorf centrifuge, 14 000 rpm, 5 min, room temperature) and subjected to GC-MS analysis.

In the two independent main experiments (1st and 2nd main experiment), Cyclohexyl Salicylate (1 μM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 20 minutes as described above. Reactions were stopped at time 0, 2.5, 5, 10 minutes and 20 minutes. As control, the test substance was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors for 0 minutes and 20 minutes and in presence of active S9 protein in absence of any cofactors for 20 minutes. Reactions were stopped and extracted as described above.

Details on estimation of bioconcentration:

The model version used was “S9spreadsheet_4202012_standardfish_consensusver-1.xlsx”. The S9 in vitro substrate depletion rate is converted to a whole-fish metabolism rate constant (Kmetab) using a number of extrapolation and scaling factors. The estimated Kmetab value is then used to refine the partitioning based BCF model prediction. A binding term, fu, is used to correct for the difference in free chemical concentration between in vivo 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 logKow-based algorithms, or 2) binding in vitro and in vivo can be assumed to be equal ( fu = 1.0). The refined BCF has been estimated using both approaches.

Type:

BCF

Value:

329 L/kg

Calculation basis:

other: Estimated using in vitro to in vivo extrapolation model.

Remarks on result:

other: Refined BCF for Cyclohexyl Salicylate, including biotransformation rate estimated (fu calculated)

Type:

BCF

Value:

144 L/kg

Calculation basis:

other: Estimated using in vitro to in vivo extrapolation model.

Remarks on result:

other: Refined BCF for Cyclohexyl Salicylate, including biotransformation rate estimated (fu = 1)

Details on results:

A rapid decrease of Cyclohexyl Salicylate within 30 minutes (86.0% decrease) and an almost complete decrease within 120 minutes (96.8% decrease) was observed in the range finding experiment with active S9 protein. In the absence of any cofactors added, a slower decrease of Cyclohexyl Salicylate was observed (8.1% within 120 minutes). A negligible decrease of Cyclohexyl Salicylate was observed in the control samples with heat inactivated protein (0.5% decrease within 120 minutes) (Appendix 2).

Thus, incubations were carried out up to 20 minutes in the two main experiments. Enzymatic turnover of Cyclohexyl Salicylate was rapid in presence of cofactors (Fig. 2). 68.3% and 69.9% decrease was observed for Cyclohexyl Salicylate in 20 minutes in two independent experiments. No to minor turnover of Cyclohexyl Salicylate was found with active S9 protein in the absence of cofactors (0% and 8.4% decrease in 20 minutes). There was no to minor decrease of Cyclohexyl Salicylate observed with inactive S9 protein (0% and 5.6% decrease in 20 minutes).

The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transform measured concentrations of parent compound as a function of time in two independent experiments: 3.42 and 3.57 ml/h/mg protein (Appendix 3).

The in vitro substrate depletion rates were used as input into the in vitro-in vivo extrapolation model to generate estimated BCF values (see table 1 in the section "Any other information on results incl. tables").

Table 1. Refined BCF estimates calculated with the in vitro–in vivo extrapolation model [1]

 

	Cyclohexyl Salicylate
Parameter	fu calc [2]	fu=1.0 [3]
Parameter: in vitro data
S9 concentration (CS9) (mg/mL)	1	1
Reaction rate (Rate) (1/h)	3.50	3.50
Input Parameter
LogKow	4.7	4.7
Calculated Parameters
Partitioning based BCF, assuming no metabolism (BCFp)	2506	2506
In vitrointrinsic clearance (CLIN VITRO,INT) (ml/h/mg protein)	4.3	4.3
In vivointrinsic clearance (CLIN VIVO,INT) (l/d/kg fish)	77.6	77.6
Scaled clearance for 10 g fish (CLIN VIVO,INT,10) (l/d/kg fish)	232.0	232.0
Corrected clearance for 10 g fish (CLIN VIVO,INT,10,CORR) (l/d/kg fish)	579.9	579.9
Hepatic clearance (CLH) (L/d/kg fish)	9.5	23.5
Whole-body metabolism rate (kMETAB) (1/d)	1.6	4.1
BCF,on a total conc basis, w/out lipid norm.(BCFTOT) (l/kg)	329	144

 

¹J. Nichols, personal communication (version: “S9spreadsheet_4202012_standardfish_consensusver-1.xlsx”), based on a previous publication from Nichols et al [2006]

²fu, plasma binding correction term; “fu calc”, hepatic clearance is calculated taking into account a theoretically calculated difference between in vitro and in vivo binding

³fu, plasma binding correction term; “fu = 1.0”, hepatic clearance is calculated assuming equa lin vitroand in vivo binding by setting fu = 1.0

Conclusions:

Metabolic turnover by trout liver S9 fractions was observed for Cyclohexyl Salicylate, indicating that Cyclohexyl Salicylate is expected to be metabolised in vivo. The capability of fish to metabolise substances to more polar components, leads to lower BCF values. Indeed, a partitioning based BCF estimate (assuming no metabolism) was reduced from 2506 L/kg to 114-329 L/Kg by incorporating in vitro depletion rates into an "in vitro - in vivo" extrapolation model. These refined BCF estimates indicate that Cyclohexyl Salicylate is expected to have a low potential for bioaccumulation.

Executive summary:

Introduction: A study was performed to assess the in vitro stability of Cyclohexyl Salicylate in fish liver S9 fractions. The method followed was a standardised assay, prevalidated by a consortium under the coordination of HESI/ILSI.

 

Experimental: Following a preliminary range finding test, Cyclohexyl Salicylate (1 µM) was incubated in triplicate with trout liver S9 fraction (1 mg/ml) for 0, 2.5, 5, 10 and 20 minutes at 12°C in two main, independent experiments. Negative controls included incubation of the test substance with heat inactivated S9 protein.

The disappearance of Cyclohexyl Salicylate as a function of time was monitored using GC-MS analysis. The rate of substrate depletion was calculated within the linear range of parent decrease and used as input into an "in vitro - in vivo" extrapolation model to generate an estimated BCF for a "standardised fish".

 

Results: Cyclohexyl Salicylate demonstrated a metabolic turnover of 68 to 70% of the starting concentration within a 20 minute exposure period. In contrast, there was no significant decrease of Cyclohexyl Salicylate with the heat inactivated S9 control.

The in vitro intrinsic clearance was calculated from the log-transform measured concentrations of parent compound as a function of time in two independent experiments: 3.42 and 3.57 ml/h/mg protein. The average in vitro intrinsic clearance rate (3.5 ml/h/mg protein) was used as input into an in vitro-in vivo extrapolation model to generate a refined BCF estimate of 144 l/kg using an assumed fu = 1.0 (i.e. no effect of differential binding to serum) and 329 l/kg assuming different binding to serum in vivo versus in vitro (fu calc).

Conclusions: Metabolic turnover by trout liver S9 fractions was observed for Cyclohexyl Salicylate, indicating that Cyclohexyl Salicylate is expected to be metabolised in vivo. The capability of fish to metabolise substances to more polar components, leads to lower BCF values. Indeed, a partitioning based BCF estimate (assuming no metabolism) was reduced from 2506 L/kg to 114-329 L/Kg by incorporating in vitro depletion rates into an "in vitro - in vivo" extrapolation model. These refined BCF estimates indicate that Cyclohexyl Salicylate is expected to have a low potential for bioaccumulation.

 

Reference 4

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Supporting information on an In vivo Fish BCF study on a structurally similar analogue.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)

Qualifier:

according to guideline

Guideline:

EU Method C.13 (Bioconcentration: Flow-through fish test)

Version / remarks:

EEc Directive 79/831, Annex V, Part C

Deviations:

no

Specific details on test material used for the study:

CYCLOHEXYL SALICYLATE Carboxyl-14C
Batch No. 92301
Specific Activity 9.41 x 106 Bq/mg (2.09 x 109 Bq/mmol), dissolved in ethanol with Specific Activity of 1.67 x 107 Bq/mL;
Radiopurity >98%

Radiolabelling:

yes

Details on sampling:

Fish were sampled from the two basins at regular intervals to measure the concentration of 14C-labelled test substance in the tissues.

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test sub-stances)
- Method: automatic dosing system adding proportionate volumes of test substance solution and dilution water to the mixing vessel; the mixing ratio of test substance concentrate and dilution water is 1:100
- Controls: not required since 14C-labelled test material was used
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): 100-times concentrates contained 0.1 mL ethanol per one concentrate
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no,

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: West Aquarium GmbH
- Age at study initiation (mean and range, SD): not given
- Length at study initiation (lenght definition, mean, range and SD): about 3 cm
- Weight at study initiation (mean and range, SD): 0.219 ± 0.05 g for fish used for lipid content determination
- Weight at termination (mean and range, SD): 0.295 ± 0.07 g for fish used for lipid content determination
- Health status: low mortality of < 1% during the last two weeks before study start
- Description of housing/holding area: test basins with 6 L volume (minimal volume 3 L), about 10 water changes per day, automatic cleaning and removal of food residues and faeces, constant temperature of 23 °C ± 1 °C

FEEDING DURING STUDY
- Food type: Altromin fish food N-1324
- Amount: about 2% of actual fish wet weight
- Frequency: automatically

ACCLIMATION
- Acclimation period: fish were delivered about one month before study start, 2 days before start the fish were placed in the test basins.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

14 d

Hardness:

Not reported

Test temperature:

Basin 1 : 23 +/- 0.13 °C
Basin 2 : 23.1 +/- 0.12 °C

pH:

Basin 1 : 7.51 +/- 0.07
Basin 2 : 7.50 +/- 0.08

Dissolved oxygen:

Basin 1 : 79.9 % of saturation +/- 8.3%
Basin 2 :79.1 % +/- 9.3%

TOC:

Not reported.

Salinity:

Not applicable.

Details on test conditions:

Details on Test Conditions
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: glass vessels, 6 L fill volume
- Aeration: continuous aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 10 renewals per day
- No. of organisms per vessel: Basin 1 contained 55 and Basin 2 40 fish
- No. of vessels per concentration (replicates): 1
- Biomass loading rate: 3 g fish wet weight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water filtered through active carbon
- Composition of test medium: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: not reported.


Uptake Phase – The study was conducted as a flow-through test at 23 °C in filtered drinking water. Fish were exposed in 6 Litre tanks. Ten volume exchanges were performed per day. Oxygen concentration was > 60% of saturation. Two exposure concentrations were performed : 1 microg/L and 10 microg/L. Fish samples were taken at regular intervals (Day0, 2, 6, 12, 20 and 28), left to drip dry, euthanised and combusted to determine total C-14 content via 14CO2. At the end of the uptake phase (Day 28), 15 fish were sampled from the 1 microg/L and extracted with ethanol to determine test material. There was a > 20% variation in the test material concentration in the test vessels during the first 2 Days of the uptake phase presumably due to strong adsorption of the test material.

Depuration Phase – Fish were transferred from the treated aqueous phase to clean water containing no test substance, and, incubated for a period of 14 Days. Fish were removed for analysis on Day 30, 33, 37 and 42. The lipid content was determined on fish from the 10 microg/L treatment level at the end of the depuration phase of the study.

Nominal and measured concentrations:

Nominal concentrations: 1 and 10 µg/L (Basin 1 and Basin 2, respectively)
Mean measured concentrations (measured with 14C activity determination): 1.05 and 8.5 µg/L.

Reference substance (positive control):

no

Lipid content:

9.35 %

Time point:

end of exposure

Lipid content:

13.92 %

Time point:

start of exposure

Conc. / dose:

>= 1 - <= 10 µg/L

Temp.:

23 °C

pH:

7.5

Type:

BCF

Value:

>= 600 - <= 900 L/kg

Basis:

whole body w.w.

Time of plateau:

5 d

Calculation basis:

other: Steady-state BCF for Parent material

Remarks:

TLC analysis of ethanol extracts of fish showed the greatest portion of the extracted radioac-tivity (ca. 50 to 75%) was the unchanged test substance. From the analytical determinations, a BCF of 600 to 900 was calculated for the parent substance.

Remarks on result:

other: Parent specific analyses.

Conc. / dose:

10 µg/L

Temp.:

23 °C

pH:

7.5

Type:

BCF

Value:

1 170 L/kg

Basis:

whole body w.w.

Time of plateau:

5 d

Calculation basis:

steady state

Remarks on result:

other: Based on Total Radioactivity

Conc. / dose:

1 µg/L

Temp.:

23 °C

pH:

7.51

Type:

BCF

Value:

1 136 L/kg

Basis:

whole body w.w.

Time of plateau:

5 d

Calculation basis:

steady state

Remarks on result:

other: Total Radioactivity

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

2.5 d

Details on kinetic parameters:

Not calculated, but the depuration half-life was clearly below 3 days in the study.

Metabolites:

Not investigated.

Results with reference substance (positive control):

Not applicable.

Validity criteria fulfilled:

yes

Remarks:

The lipid content of fish at the end of the test was slightly lower than generally acceptable.

Conclusions:

The bioconcentration factor for Cyclohexyl salicylate (analogue substance) was determined by using 14C-labelled test material. The experimentally determined values at test concentra-tions of 1 and 10 µg/L were 1136 and 1170 L/kg, respectively based on total radioactivity. Analytical determination of the test substance concentrations in ethanol extracts of fish tissue by thin layer chromatography gave BCF values ranging from 600 to 900 L/kg.

Executive summary:

The bioaccumulation potential of the radiolabelled test substance Cyclohexyl salicylate [Carboxyl 14-C] was determined under GLP in accordance with EU Method C.13 under flow-through conditions. Zebra fish (Danio rerio) of about 3 cm length and 0.2 to 0.3 g weight were used in the experiment. The lipid content of fish was about 14% at the begin of the study and decreased to 9.4% at the end of the study. This decrease was slightly above the generally acceptable decrease of 25%. Two test concentrations of nominal 1 and 10 µg/L were used in the accumulation phase over a period of 28 days. The measured time-weighted average concentrations were 1.05 and 8.5 µg/L, respectively. Fish were sampled at regular intervals, put into liquid nitrogen, weighed and measured. The animals were burned in an oxidiser and the concentration of radiolabelled substance was measured with a liquid scintillation counter. The plateau was reached quickly after a few days (less than 5). The experimentally determined BCF values obtained with the test concentrations of 1 and 10 µg/L were 1136 and 1170, respectively.

After 28 days, a 14-day depuration phase was started by placing the test fish in basins with dilution water not containing the test substance. A rapid depuration was seen and the corresponding depuration half-life was clearly less than 3 days. Analytical determination of the test substance concentrations in ethanol extracts of fish tissue by thin layer chromatography gave BCF values ranging from 600 to 900.

Overall, it can be concluded that the substance Cyclohexyl salicylate tends to rapidly bioconcentrate in the zebra fish and that a plateau is reached quickly within less than 5 days. However, also the depuration is very rapid and the depuration half-life is clearly less than 3 days. The experimentally determined bioconcentration factors are clearly below the threshold limit value of 2000 indicating bioaccumulation potential. The substance therefore should be considered as not bioaccumulative.

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.