Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

The test substance, 'Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is produced by ADM (in a closed system) in the US and imported into the EU by the further processors (downstream users). Thereafter, the ADM substance is processed by their customers/importers in a dedicated industrial setting, to produce first crude squalene and then in another process to produce pure squalene. Squalene can be marketed as such, or more usually, converted by further processors to squalane. Therefore, the CSR/exposure assessment only covers the use of the test substance, in the manufacture of other substances (e.g., crude squalene, squalane). No other uses are covered in the CSR, since the first processing of the ADM substance to crude squalene is the end use of the ADM substance.

There is no likelihood of exposure of the ADM substance as such to the environment, due to the extensive processing of the substance by the importers in closed reactors for the manufacturing of other substances. There is no evidence of residues of the ADM substance in the end product (confirmed by the customers) or in the STP as the reactors are not flushed with water but are instead sequentially batch filled with new material. Further, there is also no likelihood of exposure to the ADM substance via air, as all reactors at the customer site are closed and there is no release to air.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

20 Jan 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction from a well-known and acknowledged tool. See below under "attached background material section" for methodology and QPRF

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

REACH Guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals

Principles of method if other than guideline:

The test substance ‘Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is a UVCB constituting of 2-6% Glycerides, vegetable-oil, mono- and di-, 5-8% fatty acids, vegetable-oil; 9-15% fatty acids, vegetable-oil, Me esters; 15-55% squalene, 5-8% tocopherols, 12-20% plant sterols as well as plant sterol esters (which will hydrolyse to plant sterols) and 10-15% long chain hydrocarbons (C27 and C33 or C34). Therefore, its 96-h LC50 values were predicted for the individual substances which are representative of the major components, using SMILES as the input parameter and based on regression-based class-specific equations from the ECOSAR program (e.g., esters, phenols). The regression-based equations are based on log Kow and MW as the two dependent parameters.

Analytical monitoring:

no

Test organisms (species):

other: Fish

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 0 - ca. 3.89 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 range for all constituents (irrespective of their solubility values)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 2.08 - ca. 3.89 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 range for 4 constituents which are below water solubility; (water solubilty values ranged from 64.4 to 3.4 mg/L)

Remarks:

more lilkely to have effects at saturation

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 0 - ca. 0.012 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 range for 6 constituents which exceeds the water solubility by 1-10X; (water solubility values ranged from 3.2E-10 to 1.2E-2 mg/L)

Remarks:

not likely to have effects at saturation due to poor water solubility

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 0 - ca. 0.004 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 range for 4 constituents which exceeds the water solubility by 10X; (water solubility values ranged from 6.53E-21 to 1.13E-4 mg/L)

Remarks:

no effects at saturation (NES)

Details on results:

See below table for details on the LC50 predictions and domain evaluation

Sublethal observations / clinical signs:

Table 1: Fish 96 h LC50 predictions: Regression-based equations

Category name of constituents	Constituent names#	Concentration (% w/w)	96 h Fish LC50 (mg/L) (ECOSAR^TMv.2.0)
Glycerides, vegetable-oil	Trioctanoin	≥5 - ≤8	3.90E-03
	Tristearin		1.80E-11
	Triolein		4.40E-11
Glycerides, vegetable-oil, mono- and di-	Glycerol 1,3-dilaurate	≥2 - ≤6	3.90E-03
	Glycerol monolaurate		3.89
Fatty acids, vegetable-oil	Lauric acid	>0 - ≤2	3.34
Fatty acids, vegetable-oil, Me esters	Methyl caprylate	≥9 - ≤15	3.59
	Methyl stearate		9.20E-03
	Methyl oleate		1.20E-02
Squalene	Squalene	≥15 - ≤55	4.40E-09
Vitamin E	alpha-tocopherol	≥5 - ≤8	1.40E-05
Plant sterols and plant sterol esters (which will hydrolyse to plan sterols)	beta-sitosterol (as sterol)	≥12 - ≤20	4.60E-05
	beta-sitosterol (as sterol ester)	≥3 - ≤6
Long chain hydrocarbons (C27 and C33 or C34)	2,6,10,15,19,23‐hexamethyltetracosane	≥10 - ≤15	1.60E-09
Heptane	Heptane	≥2 - ≤7	2.08

#Representative structures for each category of constituents has been considered for the QSAR modelling

Table 2: Domain evaluation

Constituent names#	ECOSAR^TMclasses	MW (g/mol)	MW cut-off (g/mol)	Log Kow (est / exp)	Log Kow cut-off	Water solubility (mg/L)	No effects at saturation (NES) – Yes/No	Domain evaluation
Trioctanoin	Esters	470.7	1000	9.2	5	1.13E-04	Yes	General (ID); MW (ID), Kow (OD)
Tristearin	Esters	891.51	1000	23.94	5	6.53E-21	Yes	General (ID); MW (ID), Kow (OD)
Triolein	Esters	885.46	1000	23.29	5	3.17E-19	Yes	General (ID); MW (ID), Kow (OD)
Glycerol 1,3-dilaurate	Esters	456.71	1000	9.18	5	6.33E-05	Yes	General (ID); MW (ID), Kow (OD)
Glycerol monolaurate	Esters	274.4	1000	3.67	5	12.67	No	General (ID); MW (ID), Kow (ID)
Lauric acid	Neutral organics	200.32	1000	4.6	5	4.81	No	General (ID); MW (ID), Kow (ID)
Methyl caprylate	Esters	158.24	1000	3.32	5	64.40	No	General (ID); MW (ID), Kow (ID)
Methyl stearate	Esters	298.51	1000	8.35	5	1.20E-03	May be NES or may have effects	General (ID); MW (ID), Kow (OD)
Methyl oleate	Esters	296.5	1000	7.45	5	1.20E-02	May be NES or may have effects	General (ID); MW (ID), Kow (OD)
Squalene	Neutral organics	410.73	1000	14.12	5	1.05E-09	May be NES or may have effects	General (ID); MW (ID), Kow (OD)
alpha-tocopherol	Phenols	416.69	1000	11.63	7	6.23E-06	May be NES or may have effects	General (ID); MW (ID), Kow (OD)
beta-sitosterol (as sterol)	Neutral organics	414.72	1000	9.65	5	1.23E-05	May be NES or may have effects	General (ID); MW (ID), Kow (OD)
beta-sitosterol (as sterol ester)	Neutral organics
2,6,10,15,19,23‐hexamethyltetracosane	Neutral organics	422.83	1000	14.63	5	3.20E-10	May be NES or may have effects	General (ID); MW (ID), Kow (OD)

MW: molecular weight; Kow: partition coefficient; ID: In domain; OD: Out of domain

#Representative structures for each category of constituents has been considered for the QSAR modelling

Table 3: Number of compounds (N) in training set andR2 (correlation or determination coefficient)

Category name of constituents	Constituent names#	ECOSAR^TMclasses	N*	R2 for fish 96 h LC50 predictions
Glycerides, vegetable-oil	Trioctanoin	Esters	102 + 13	0.7937
	Tristearin	Esters	102 + 13	0.7937
	Triolein	Esters	102 + 13	0.7937
Glycerides, vegetable-oil, mono- and di-	Glycerol 1,3-dilaurate	Esters	102 + 13	0.7937
	Glycerol monolaurate	Esters	102 + 13	0.7937
Fatty acids, vegetable-oil	Lauric acid	Neutral organics	296 + 55	0.878
Fatty acids, vegetable-oil, Me esters	Methyl caprylate	Esters	102 + 13	0.7937
	Methyl stearate	Esters	102 + 13	0.7937
	Methyl oleate	Esters	102 + 13	0.7937
Squalene	Squalene	Neutral organics	296 + 55	0.7937
Vitamin E	alpha-tocopherol	Phenols	188 + 10	0.868
Plant sterols and plant sterol esters (which will hydrolyse to plan sterols)	beta-sitosterol (as sterol)	Neutral organics	296 + 55	0.7937
	beta-sitosterol (as sterol ester)	Neutral organics	296 + 55	0.7937
Long chain hydrocarbons (C27 and C33 or C34)	2,6,10,15,19,23‐hexamethyltetracosane	Neutral organics	296 + 55	0.7937
Heptane	Heptane	Neutral organics	296 + 55	0.7937

* N = x + y where “x” equals the number of studies used in actual equation development and “y” equals (1) Log Kow cut-off and/or (2) chemicals listed under the SAR Data not included in Regression Equation

#Representative structures for each category of constituents has been considered for the QSAR modelling

Considering that the number of training set compounds for each of the chosenECOSAR^TMclasses and R2 >0.7 are not too low, the predictions are considered to be reliable with moderate confidence.

Validity criteria fulfilled:

not applicable

Conclusions:

Therefore, given the poor to low water solubility of the different constituents together with their predicted 96-h LC50 values, the test substance can be overall expected to have either ‘no effects at saturation’ or a low acute aquatic toxicity potential with LC50 exceeding 1 mg/L.

Executive summary:

The fish 96-h LC50 value of test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ was predicted using the class specific regression-based equations from ECOSAR Class Program v2.0. Since the test substance is a UVCB constituting of 2-6% Glycerides, vegetable-oil, mono- and di-, 5-8% fatty acids, vegetable-oil; 9-15% fatty acids, vegetable-oil, Me esters; 15-55% squalene, 5-8% tocopherols, 12-20% plant sterols as well as plant sterol esters (which will hydrolyse to plan sterols) and 10-15% long chain hydrocarbons (C27 and C33 or C34), the 96-h LC50 values were predicted for substances, which are representative of the major components. SMILES code was used as the input parameter. Using the ECOSAR class specific regression-based equations, the 96-h LC50 values for the 14 structures ranged from 1.8E-11 to 3.89 mg/L. Out of the 14 constituents/structures, the LC50 values for 10 of them were predicted to exceed their water solubility values, where 4 of them exceed by 10X times indicating that they will have no effects at saturation (NES). The 96-h LC50 values of these 10 constituents ranged from 1.8E-11 to 1.2E-2 mg/L and their corresponding water solubility values ranged from 6.53E-21 to 1.2E-2 mg/L, indicating an overall likelihood of no effects at saturation due to poor water solubility. Lastly, the 96-h LC50 values of the remaining 4 constituents (which were below their corresponding water solubility) ranged from 2.08 to 3.89 mg/L, indicating a low acute aquatic potential. Therefore, given the poor to low water solubility of the different constituents together with their predicted 96-h LC50 values, the test substance can be overall expected to have either ‘no effects at saturation’ or a low acute aquatic toxicity potential with LC50 exceeding 1 mg/L. On comparing with the general and molecular descriptors for the domain evaluation, all constituents were found to meet the MW domain criteria, however 10 out of 14 constituents did not meet the log Kow cut offs as defined in the ECOSARTM help manual. Therefore, the predictions can be overall considered to be accurate with moderate uncertainty.

Description of key information

Overall, based on the predicted 96-h LC50 values for fish combined with the lack of environmental exposure suggests that the test substance is not expected to pose any concern to the aquatic organisms.

Additional information

According to Annex XI Section 1.2 (weight of evidence), the study does not need to be conducted because there is no direct or indirect exposure of the aquatic compartment due to the intermediate use of the substance. In addition, there are mitigating factors indicating that aquatic toxicity is unlikely to occur, as the substance is poorly soluble in water. The test substance is manufactured outside the EU and the importers thereafter extensively process it at dedicated sites in closed reactors for the manufacturing of other substances (e.g., squalene or squalane). There is no evidence of residues of the substance in the end product or in sewage treatment plants as the reactors are not flushed with water but are instead sequentially batch filled with new material. Further, there is no likelihood of exposure to the substance via air, as all reactors at the customer site are closed and there is no release to air (see Chapters 9 & 10 for further exposure details). 

Nevertheless, the fish 96-h LC50 value of test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ was predicted using the class specific regression-based equations from ECOSAR Class Program v2.0. Since the test substance is a UVCB constituting of 2-6% Glycerides, vegetable-oil, mono- and di-, 5-8% fatty acids, vegetable-oil; 9-15% fatty acids, vegetable-oil, Me esters; 15-55% squalene, 5-8% tocopherols, 12-20% plant sterols as well as plant sterol esters (which will hydrolyse to plant sterols) and 10-15% long chain hydrocarbons (C27 and C33 or C34), the 96-h LC50 values were predicted for substances, which are representative of the major components. SMILES code was used as the input parameter. Using the ECOSAR class specific regression-based equations, the 96-h LC50 values for the 14 structures ranged from 1.8E-11 to 3.89 mg/L. Out of the 14 constituents/structures, the LC50 values for 10 of them were predicted to exceed their water solubility values, where 4 of them exceed by 10X times indicating that they will have no effects at saturation (NES). The 96-h LC50 values of these 10 constituents ranged from 1.8E-11 to 1.2E-2 mg/L and their corresponding water solubility values ranged from 6.53E-21 to 1.2E-2 mg/L, indicating an overall likelihood of no effects at saturation due to poor water solubility. Lastly, the 96-h LC50 values of the remaining 4 constituents (which were below their corresponding water solubility) ranged from 2.08 to 3.89 mg/L, indicating a low acute aquatic potential. Therefore, given the poor to low water solubility of the different constituents together with their predicted 96-h LC50 values, the test substance can be overall expected to have either ‘no effects at saturation’ or a low acute aquatic toxicity potential with LC50 exceeding 1 mg/L. On comparing with the general and molecular descriptors for the domain evaluation, all constituents were found to meet the MW domain criteria, however 10 out of 14 constituents did not meet the log Kow cut offs as defined in the ECOSARTM help manual. Therefore, the predictions can be overall considered to be accurate with moderate uncertainty.

Overall, based on the predicted 96-h LC50 values for fish combined with the lack of environmental exposure suggests that the test substance is not expected to pose any concern to the aquatic organisms.