Sophorolipids: fermentation products of glucose and rapeseed-oil fatty acids methyl esters with yeast Candida Bombicola

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 22.05.2019 and 27.09.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The method has recently been adopted as a new test guideline by the OECD; TG No. 249 Fish Cell Line Acute Toxicity: The RTgill-W1 cell line assay (adopted 14 June, 2021).

Qualifier:

according to guideline

Guideline:

other: International Organization for Standardization (ISO) : Water Quality - Determination of Acute Toxicity of Water Samples and Chemicals to a Fish Gill Cell Line (RTgill-W1), ISO21115.

Qualifier:

equivalent or similar to guideline

Guideline:

other: The method has recently been adopted as a new test guideline by the OECD - TG No. 249 Fish Cell Line Acute Toxicity: The RTgill-W1 cell line assay

Version / remarks:

Adopted 14 June 2021

GLP compliance:

no

Remarks:

Conducted in compliance with the standard operating procedure version CS-02 of the CEFIC-LRI funded CEllSens validation study on the replacement of acute fish toxicity studies by the RT-gill W1 assay, which is compatible with the ISO 21115:2019.

Analytical monitoring:

no

Remarks:

Quantification of Sopholiance S was not possible due to a poor GC elution profile and a non-linear calibration. Therefore, all results are based on nominal concentrations.

Vehicle:

yes

Remarks:

DMSO - final concentration in the test medium was 0.5%

Details on test solutions:

Test Plate Dosing and Incubation
The test chemical was dissolved to a final concentration of 25.6 mg/mL in Dimethylsulfoxide (DMSO).
Serial half dilutions in DMSO were prepared resulting in final concentrations of 25.6, 12.8, 6.4, 3.2,
1.6 and 0.8 mg/mL. From each DMSO solution, a dosing solution in medium was prepared by adding
50 μL to 9.95 mL of exposure medium L15/ex (final nominal test chemical concentrations: 128, 64, 32,
16, 8, 4 mg/L). Final DMSO concentration in the test medium was 0.5% for both experiments.
L15 medium was removed completely from each well of the 24-well plate which had been seeded with
the cells 24 h earlier. Cells were washed once with 1 mL exposure medium L15/ex and 2 mL dosing
solution was added to each well. Solvent controls received 2 mL L15/ex containing 0.5% DMSO. The
plate was then covered with a plastic sealing foil.
The plate was incubated for 24 h in a BINDER Model KT 115 refrigerated incubator with
thermoelectric cooling, maintained at 19°C with normal air (no CO2 enrichment) and 40% ventilation
rate.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test species = RT-gill W1 cell line from rainbow trout.
The rainbow trout (Oncorhynchus mykiss) RTgill-W1 cell line was obtained from the Swiss center for aquatic research EAWAG (Dübendorf, Switzerland). The cells were routinely cultured in 75 cm2 cell culture flasks containing Leibovitz (L15) medium without phenol red supplemented with Glutamine (Supplier no. 21083; Gibco-Invitrogen; Basel, Switzerland), 5% foetal bovine serum (FBS) and 0.05 mg/mL Gentamicin. The cells were split each week once with a 1:1 split. The flasks are incubated in a BINDER Model KT 115 refrigerated incubator with thermoelectric cooling, maintained at 19°C with ambient air (no CO2 enrichment) and 40% ventilation rate.

Test type:

static

Water media type:

other: The exposure medium was Leibovitz L15/ex. The preparation is described in Appendix C of the study report.

Limit test:

no

Total exposure duration:

24 h

Test temperature:

19°C

Nominal and measured concentrations:

-Two experiments were performed.
- Both experiments were conducted at 128, 64, 32, 16, 8, 4 mg/L based on nominal concentrations. Each concentration was tested in triplicate.
- A solvent control (0.5% DMSO; L15/ex) in triplicate in presence of cells was included in both tests and compared to a cell control without solvent.
- Additionally, one well without cells and the highest test chemical concentration and one well without cells and no test chemical were prepared as control to correct for background
fluorescence for the cell viability test.

Details on test conditions:

Preparation of Test Plates with RTgill-W1 Cells:
For exposure to the test chemical, confluent cells (80 - 90% confluence) were gently washed with
2 mL Versene (Supplier no. 15040, Gibco-Invitrogen). Cells were then detached with 0.7 mL trypsine solution (0.25% in phosphate buffered saline (PBS) w/o calcium and magnesium) and trypsination was stopped by adding 10 mL L15 medium supplemented with FBS. Cell clumps were dissolved by gently pipetting the solution several times up and down and the cell suspension was centrifuged for 3 min at 1000 rpm. The cell pellet was suspended in 10 mL L15 medium with FBS and counted in a Neubauer chamber. Cell density was adjusted to 350 000 cells / mL and 1 mL was seeded in each well of a 24-well plate, except for two wells without cells to correct for background fluorescence. After 24 h of attachment, the toxicity testing was initiated by adding the test chemical dilutions in exposure medium.

Test Plate Dosing and Incubation:
The test chemical was dissolved to a final concentration of 25.6 mg/mL in Dimethylsulfoxide (DMSO). Serial half dilutions in DMSO were prepared resulting in final concentrations of 25.6, 12.8, 6.4, 3.2, 1.6 and 0.8 mg/mL. From each DMSO solution, a dosing solution in medium was prepared by adding 50 μL to 9.95 mL of exposure medium L15/ex (final nominal test chemical concentrations: 128, 64, 32, 16, 8, 4 mg/L). Final DMSO concentration in the test medium was 0.5% for both experiments. L15 medium was removed completely from each well of the 24-well plate which had been seeded with the cells 24 h earlier. Cells were washed once with 1 mL exposure medium L15/ex and 2 mL dosing solution was added to each well. Solvent controls received 2 mL L15/ex containing 0.5% DMSO. The plate was then covered with a plastic sealing foil.
The plate was incubated for 24 h in a BINDER Model KT 115 refrigerated incubator with
thermoelectric cooling, maintained at 19°C with normal air (no CO2 enrichment) and 40% ventilation rate.

Test Plate Sampling and viability determination:
After 24 h incubation, the dosing solution was discarded and each well was washed with 1 mL of
PBS. The washing solution was discarded and to each well 400 μL PrestoBlue® working solution
(PrestoBlue® reagent diluted 10-fold in PBS) was added and the cells were incubated at 19°C for
30 min in the dark. The PrestoBlue® fluorescence was then measured at excitation of 560 nm and
emission of 590 nm. All measurements were performed on a Synergy H1 Hybrid Reader (BioTek) in the bottom read mode with read height of 4.40 mm and with a gain of 104.

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline (DCA) is regularly tested as reference chemical and EC50 compared to the results of the international ring trial to validate the performance of the assay. A summary of the results is given in Appendix A of the full study report.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Predicted in vivo acute toxicity (experiment 2)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

12.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Cytotoxicity

Remarks on result:

other: Experiment 2

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Predicted in vivo acute toxicity (experiment 1)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

13.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Cytotoxicity

Remarks on result:

other: Experiment 1

Details on results:

Extrapolation from in vitro to in vivo:
In order to predict a fish in vivo LC50 value from the in vitro EC50, a detailed benchmarking study on 38 fragrance chemicals was performed (Summary appended to the study report). This led to a predictive regression equation based on the EC50 nominal determined with PrestoBlue® (PB):

Log LC50fish in vivo = (1.11 × log EC50 PrestoBlue® gill nominal) – 0.72

This regression equation was used to predict an in vivo LC50.

As proposed in the benchmarking study on 38 fragrance chemicals, effect calculations (e.g. EC50) should be based on nominal concentrations instead of arithmetic mean measured
concentrations in the case of a lack of appropriate analytical methods for the test chemical, such is the case of Sopholiance S. The predicted LC50 based on in vitro nominal EC50 has been shown to have a comparable reliability to the regression based on in vitro mean measured EC50 – see reference [7] and Appendix D in the full study report for further details.

Results with reference substance (positive control):

The EC50 for DCA determined in two independent experiments was 38.55 and 49.74 mg/L, respectively, which is fulfilling the acceptance criteria for this positive control. Based on the results of the international validation study, the acceptance range for DCA was set as 43.6 mg/L +/- 6.1 mg/L for metabolic activity (2.5 SD range: 28.4-58.9 mg/L).

Reported statistics and error estimates:

The median of the under-/over-prediction (fold difference between LC50 predicted by equation 2 in Appendix D and measured in vivo LC50) was 1.4-fold for the series of 37 fragrance substances tested [7] which is considered to be well within the variation in LC50 if a chemical is tested in different fish species. Furthermore, a factor of 1.4-fold is considered to be also well within the end-point variation for intra- and inter-laboratory testing using the same species.

Sublethal observations / clinical signs:

Cytotoxicity test:

The three acceptance criteria discussed by Fischer et al. were fulfilled: (i) The DMSO versus solventfree control wells did not differ in raw fluorescence values by more than 10%. 5.4 and 8.4 % reduction in control without solvent were measured in experiment 1 and 2, respectively (see Appendix E in Study Report); (ii) the raw fluorescence values in the cell-free control well containing the highest test chemical concentration did not vary by more than 20% from the cell-free control well containing the exposure medium only (7 and 20 % variation measured in experiment 1 and 2, respectively, Appendix E in Study Report); (iii) EC50 values of the positive control (DCA) were within the given range based on the international validation study.

Fischer, M., et al., Repeatability and reproducibility of the RTgill-W1 cell line assay for predicting fish acute toxicity. Toxicological Sciences, 2019. 169(2): p. 353-364.

Validity criteria fulfilled:

yes

Remarks:

The three acceptance criteria discussed by Fischer et al. were fulfilled (see other information on results, incl tables).

Conclusions:

The EC50 values based on nominal concentrations in the RTgill-W1 cell assay were 13.42 mg/L and 12.10 mg/L in the first and second experiment, respectively (average 12.76 mg/L). Based on a regression equation developed with 37 fragrance molecules an in vivo LC50 of 3.40 mg/L and
3.03 mg/L, respectively, is predicted (mean LC50: 3.22 mg/L).

Description of key information

The registered substance is used in Cosmetics applications. In Europe, the testing of Cosmetics ingredients on vertebrates is prohibited according to Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009, Under Article 18. Therefore, a non-animal alternative has been used to address the acute fish endpoint.    

An RTgill-W1 cell line assay was performed on Sopholiance S to predict the fish acute toxicity. This non-animal alternative method has been validated and published by the International Organization for Standardization (ISO) under the Water Quality Test Guideline ISO 21115, Determination of Acute Toxicity of Water Samples and Chemicals to a Fish Gill Cell Line (RTgill-W1), and adopted as a new test guideline by the OECD; TG No. 249 Fish Cell Line Acute Toxicity: The RTgill-W1 cell line assay (adopted 14 June, 2021). In addition, and, within our own laboratories, the method has been validated in the official laboratory ring-trial and, further validated and implemented in an internal study on 38 fragrance ingredients of varying physico-chemical properties and diverse chemistries. This work has been subsequently published by Natsch et al. (2018), Environmental Toxicology and Chemistry, 37 (3), pgs. 931 - 941.  

A fish in vivo LC50 value is predicted from the in vitro EC50 by application of an appropriate regression equation. The mean predicted LC50 value for the regsitered substance was 3.22 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

3.22 mg/L

Additional information

A Fish Gill Cell Line (RTgill-W1) assay was performed on Sopholiance S. The objective of the study was to determine the effects of Sopholiance S on the viability of the RTgill-W1 cell line to predict the fish acute toxicity. This cell line from rainbow trout (Oncorhynchus mykiss) can be used to assess toxicity of chemicals to the gills which are the organ of fish in most direct contact to environmental chemicals. The close correlation of this in vitro assay has already been demonstrated for predicting acute fish toxicity of chemicals with a narcotic mode of action [Tanneberger, K., et al, 2013]. Further, a benchmarking study has been performed on 38 fragrance chemicals, covering a broad range of physico-chemical properties and diverse chemistries, and, possessing good quality in vivo fish toxicity studies [Natsch, A., et al., 2018].

The RTgill-W1 cells were seeded in 24-well plates and exposed in minimal medium to different Sopholiance S concentrations and controls without chemical in triplicate for 24 h at 19°C. Cell viability was determined at 24 h using metabolic activity as endpoint (PrestoBlue® assay). Sopholiance S consists of multiple constituents and no reliable quantification of the test substance was possible due to a poor GC elution profile and non-linear calibration. Therefore, all results have been based on nominal concentrations.

Two experiments with the RTgill-W1 assay were performed using six different test concentrations; 128, 64, 32, 16, 8, 4 mg/L in both experiments. Each concentration was tested in triplicate. The EC50 value based on nominal concentrations was 13.42 mg/L in the first experiment and 12.10 mg/L in the second experiment.

In order to predict a fish in vivo LC50 value from the in vitro EC50, a detailed benchmarking study on 38 fragrance chemicals was performed [Natsch, A., et al., 2018]. This led to a predictive regression equation based on the EC50 nominal determined with PrestoBlue® (PB):

Log LC50fish in vivo = (1.11 × log EC50 PrestoBlue® gill nominal) – 0.72

The predicted LC50 based on in vitro nominal EC50 has a comparable reliability to the regression based on in vitro mean measured EC50 [Natsch, A., et al., 2018]. The median of the under-/over-prediction (fold difference between LC50 predicted and measured in vivo LC50) was 1.4-fold for the series of  fragrance substances tested which is considered to be well within the variation in LC50 if a chemical is tested in different fish species. Furthermore, a factor of 1.4-fold is considered to be also well within the end-point variation for intra- and inter-laboratory testing using the same species. Thus for chemicals where no appropriate analytical methods are available, the above equation based on nominal concentrations can be equally used.

Given that no reliable quantification of Sopholiance S was possible, the above regression equation was used to predict an in vivo LC50 from the in vitro EC50 values of 13.42 (Experiment 1) and 12.10 mg/ (experiment 2). The predicted in vivo LC50 values are 3.40 mg/L and 3.03 mg/L, respectively, (mean LC50: 3.22 mg/L).

 

REFERENCES 

Tanneberger, K., et al., Predicting fish acute toxicity using a fish gill cell line-based toxicity assay. Environ. Sci. Technol., 2013. 47(2): p. 1110-9.

Natsch, A., et al., Accurate prediction of acute fish toxicity of fragrance chemicals with the RTgill-W1 cell assay. Environ. Toxicol. Chem., 2018. 37(3): p. 931-941.