Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Remarks:

Predicted from an in vitro study

Adequacy of study:

weight of evidence

Study period:

06 August 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test guideline is currently under evaluation

Qualifier:

equivalent or similar to guideline

Guideline:

other: New OECD TG No. 249: Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay

Version / remarks:

Adopted 14 June 2021

Principles of method if other than guideline:

RTgill-W1 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].

GLP compliance:

no

Remarks:

Testing done in internal lab

Analytical monitoring:

yes

Details on sampling:

Samples were taken and analysed from three replicates at each concentration both at the start and at the end of the exposure period of each well to determine the initial (at T0h) and final concentration (at T24h) of test item in the wells of the 24-well plate.
Shortly after addition of the medium containing the test chemical to the cells in the 24-well plate (T0h) 0.5 mL of the test medium was removed from each test well and diluted with 0.5 mL L15/ex. At T24h, 1 mL samples from the test wells were directly analysed.

Vehicle:

yes

Remarks:

0.5% DMSO

Details on test solutions:

One experiment was conducted at 256, 128, 64, 32, 16, 8 mg/L based on nominal concentrations (57.68, 47.19, 29.69, 15.60, 7.44, 3.83 mg/L based on mean measured concentrations). Each concentration was tested in triplicate. A solvent control (0.5% DMSO; L15/ex) in triplicate in presence of cells was included 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.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

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 1:2 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.

Total exposure duration:

24 h

Test temperature:

19 °C

Nominal and measured concentrations:

Nominal concentrations: 256, 128, 64, 32, 16, 8 mg/L
Mean measured concentrations: 57.68, 47.19, 29.69, 15.60, 7.44, 3.83

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline (DCA)

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

27.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Cytotoxicity

Remarks on result:

other: 95% confidence limits: 22.55 - 32.49

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

10.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Predicted in vivo acute toxicity

Sublethal observations / clinical signs:

Chemical Analysis

 

Test item was quantified based on the peak area of the three isomers. Calibrations were prepared based on the nominal concentrations used in the cell culture plates (8 – 256 mg/L). The limit of quantification (LOQ) for Test item in medium using this method was below the lowest calibration standard (8 mg/L). Since the measured concentrations of Test item declined below the lowest nominal test concentrations at 24 h incubation, concentrations at T24h were extrapolated from the calibration curves. The initial concentration (T0h) of the lowest exposure level was also extrapolated.

 

In an independent experiment the linearity of the calibration curve < 8 mg/L was evaluated using a broader range of calibration standards (0.25 – 256 mg/L). Similar results were obtained for all concentrations which were < 8 mg/L (Appendix H), thus demonstrating strong linearity in the response of the GC-FID to Test item and validity of the extrapolations to the lower concentrations. The original values were used instead of the recalculated values since these were not measured in parallel (i.e. in the same GC run) as the gill cell samples, but some weeks later and no samples from the gill cells were left for reanalysis. Measured concentrations of the test samples ranged from 6.74 to 98.04 mg/L at T0h and from 0.93 to 17.32 mg/L at T24h.

 

Table 6.1.1/1. Measured exposure concentrations of Test item.

Nominal concentrations (mg/L)	Measured concentrations (mg/L)^1
	0 h	24 h	Mean
256	98.04	17.32	57.68
128	82.12	12.26	47.19
64	51.47	7.91	29.69
32	27.37	3.84	15.60
16	12.94	1.95	7.44
8	6.74	0.93	3.83

¹Measured concentrations < 8 mg/L should be considered as approximate values, since they were outside the calibration range of this experiment and were extrapolated from the calibration curve. These concentrations were re-calculated using an additional calibration curve (0.25 – 256 mg/L) and were similar to the extrapolated values.

 

Cytotoxicity test

 

The three acceptance criteria discussed by Fischer et al. were fulfilled: (i) The DMSO versus solvent free control wells did not differ in raw fluorescence values by more than 10 % (7.5 % variation measured; (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 (2.7 % variation measured); (iii) EC50 values of the positive control (DCA) were within the given range based on the international validation study.

 

No significant (≤ 10 %) dose related cytotoxicity occurred at the three lowest test concentrations (nominal concentrations 8 - 32 mg/L) at 24 h. The NOEC was thus considered to be 15.60 mg/L (nominal concentration 32 mg/L). The EC50 value based on mean measured concentration was 27.06 mg/L. Based on the regression equation, an in vivo LC50 of 10.7 mg/L is predicted.

 

Table 6.1.1/2. Detailed cell viability data (% viability as compared to solvent control using the PrestoBlue® assay).

Nominal concentration	256 mg/L	128 mg/L	64 mg/L	32 mg/L	16 mg/L	8 mg/L
Mean measured concentration	57.7 mg/L	47.2 mg/L	29.7 mg/L	15.6 mg/L	7.4 mg/L	3.8 mg/L
Rep 1 (% viability)	4.2	3.7	15.2	96.2	107.7	105.6
Rep 2 (% viability)	3.3	3.0	6.1	100.7	107.4	104.0
Rep 3 (% viability)	3.9	2.6	51.5	100.8	94.2	95.2
Average (% viability)	3.8	3.1	24.3	99.2	103.1	101.6
Standard deviation	0.5	0.6	24.0	2.6	7.7	5.6
Coefficient of variation (%)	12.1	19.1	98.8	2.7	7.5	5.6

 

Conclusions:

The EC50 value based on mean measured concentrations in the RTgill-W1 cell assay was 27.06mg/L. Based on a regression equation developed with 37 fragrance molecules an in vivo LC50 of 10.70 mg/L is predicted.

The median of the under-/over-prediction (fold difference between LC50 predicted by equation and measured in vivo LC50) was 1.5-fold for the series of 37 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 1.5-fold is considered to be also well within the end-point variation for intra- and inter-laboratory testing using the same species.

Executive summary:

The study determined the effects of test substance 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.

 

Furthermore, 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. The regression equation resulting from this study was used to predict the acute fish toxicity for test substance.

 

The RTgill-W1 cells were seeded in 24-well plates and exposed in minimal medium to different test substance concentrations and controls without chemical in triplicate for 24 h at 19°C. GC-analysis of the test chemical concentrations in media was conducted at the start (0 h) and the end (24 h) of exposure. Cell viability was determined at 24 h using metabolic activity as endpoint (PrestoBlue® assay).

 

One experiment with the RTgill-W1 assay was performed using six different test concentrations. Concentrations ranged from 8-256 mg/L based on nominal concentrations (measured concentrations ranging from 6.74 to 98.04 mg/L at the start and 0.93 to 17.32 mg/L at the end of the exposure).

 

The mean measured test chemical concentrations were 57.68, 47.19, 29.69, 15.60, 7.44 and 3.83 mg/L (nominal concentrations 256, 128, 64, 32, 16, 8 mg/L). The effect concentrations (EC50) are based on mean measured concentrations.

 

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 this report). This led to a predictive regression equation based on the EC50 determined with PrestoBlue® (PB):

 

Log LC50 fish in vivo= (0.97 × log EC50 PB mean measured) - 0.36

 

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

 

The EC50 values and the corresponding NOEC and LOEC values are shown in the following Table, along with the extrapolated in vivo LC50 values.

	Effect concentrations expressed as mean measured concentration (mg/L)
	Cytotoxicity 24 h (mg/L)	Predicted in vivo acute toxicity (LC50) (mg/L)
EC50	27.06 (22.55 – 32.49)^1mg/L	10.70
LOEC	29.69 mg/L	n.a^.2
NOEC	15.60 mg/L	n.a.

¹Values in brackets are 95% confidence limits

²n.a. not applicable

 

The concentrations at which no significant (≤ 10%) dose related cytotoxicity occurred were ≤ 15.60 mg/L (nominal 32 mg/L). The NOEC was thus considered to be 15.60 mg/L. After 24 hours, the EC50 value based on mean measured concentration was 27.06 mg/L. Based on the regression equation derived from a training set of 37 fragrance molecule, an in vivo LC50 of 10.70 mg/L is predicted.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 June - 10 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Inspected on: 07-08 March 2018; Signed on: 28 May 2018

Analytical monitoring:

yes

Details on sampling:

Test solution specimens from an ecotoxicology study (ECT Study No:20DV1FY) conducted at ECT Oekotoxikologie GmbH, Flörsheim/Main, Germany were sent frozen to the analytical test site. After receipt all samples were stored deep frozen (≤ -18 °C) until sample preparation and analysis was performed

Test medium samples (20 mL each) without any sample stabilisation were shipped deep-frozen to the analytical test site.

The test solution specimens were taken out of the freezer storage. Before defrosting, 20% toluene was pipetted into the sample vessels (e.g. 4 mL toluene to a water sample of approx. 20 mL). The samples were then defrosted, homogenised for at least 1 min on a Vortex mixer. After phase separation 1 mL of the upper organic phase was transferred into GC vials and used directly for analysis by GC-MS. If necessary, samples were diluted with toluene to achieve final concentrations falling within the calibrated linear range of the detector response.

Details on test solutions:

A set of two stock solutions at a nominal concentration of 3.00 mg/L were prepared by dissolving the test item in test medium (section 13.4) at each time of preparation (day 0 and 2). Therefore, 16.44–16.93 mg of the test item was dissolved in 5482–5661 mL of test medium in a completely filled and closed preparation beaker. These stock solutions were stirred at 500 rpm on a magnetic stirrer for 22–22.75 hours at ambient temperature in the dark. After that, the stock solutions were not stirred for 30–45 minutes. The stock solutions were visually examined for undissolved/particulate matter and no un-dissolved test material was observed (e.g., forming a surface film), so it can be assumed that the test item was completely dissolved. Thus, the stock solutions were used directly to prepare the desired test solutions for pre-conditioning of the test vessel and for the exposure. The test vessels were pre-conditioned for 75–90 minutes. Thereafter, the test solutions were discarded and the second set of stock/test solution was transferred to the test vessels used for the exposure of the test organisms.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

− Fish Species: Danio rerio (Zebrafish)
− Supplier: stock culture (ECT)
− Spawning date: May 2020
− Material of stock vessel: glass
− Amount of water per stock vessel: approximately 27 L
− Depth of water in the stock vessel: approximately 20 cm
− Estimated number of fish kept as a stock per holding vessel (batch size): 200
− Length of fish: 0.89–1.43 cm, mean: 1.18 cm (assessed from a representative subsample consisting of 10 fish, 7 days before start of exposure)
− Body weight of fish: range: 0.0185–0.0300 g/fish, mean: 0.02407 g/fish (assessed from the control fish at end of the exposure period)
− Fish loading: 0.178 g/L (assessed from the control at end of the exposure period)
− Water: modified reconstituted water, see section 13.4
− Renewal of water: once per week
− Temperature of water: 23±2 C; 23.5–24.9°C (during 7 days before start of exposure)
− Oxygen content of water: 99% (14 days before start of exposure)
− Food: TetraMin® and Artemia nauplii
− Feeding frequency: daily ration fed in 3 portions on workdays and one portion on weekend, ad libitum, no feeding on the day before start of exposure
− Photoperiod: 14 h light / 10 h dark
− Light intensity: 100–1000 lx
− Mortalities during the period of 11 d before start of the test (<5%): 0.5%

Test type:

semi-static

Water media type:

other: Reconstituted water according to OECD 203

Remarks:

Modified

Limit test:

yes

Total exposure duration:

96 h

Hardness:

164 mg/L CaCO3

Test temperature:

21–25 °C, constant within the range of ±1 °C

pH:

7.6

Dissolved oxygen:

8.5–8.9 mg/L

Conductivity:

806 μS/cm2

Nominal and measured concentrations:

Nominal concentration: 3.00 mg/L
Measured concenntration: 2.56 mg/L

Details on test conditions:

− Volume of test solution per test vessel used for pre-conditioning: day 0: 4.625 L (C1); day 2: 4.612 L (C1)
− Volume of test solution per test vessel: day 0: 4.611 L (C0), 4.629 L (C1); day 2: 4.644 L (C0), 4.635 L (C1)
− Depth of test solution in the test vessels: 14.5 cm, the headspace between water surface and the glass lid of the test vessel was approximately 0.5 cm
− Number of fish per test vessel: 7
− Length of fish: 0.89–1.43 cm, mean: 1.18 cm (assessed from a representative subsample consisting of 10 fish, 7 days before start of exposure); 1.4–1.6 cm, mean: 1.50 cm (assessed from the control and the treated fish (limit concentration) at end of the exposure period)
− Body weight of fish: range: 0.0182–0.0300 g/fish, mean: 0.0236 g/fish (assessed from the control and the treated fish (limit concentration) fish at end of the exposure period); range: 0.0185–0.0300 g/fish, mean: 0.0241 g/fish (assessed from the control fish at end of the exposure period); range: 0.0182–0.0279 g/fish, mean: 0.0231 g/fish (assessed from the treated fish (limit concentration) at end of the exposure period)
− Fish loading: 0.0363–0.0366 g/L (assessed from the control fish at end of the exposure period)
− Renewal of test solution and control water during the test period: once after 48 hours
− Feeding: none
− Photoperiod: 14 h light / 10 h dark
− Light intensity: 540–1000 lx; measured: 812, 826 lx, (n = 2)
− Temperature: 22.5–23.5°C (daily manual measurement, n = 12); 21.9–23.2°C; automatic measurement; n = 199)
− Aeration: permanent (slightly aeration, approx. one bubble per second)

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 2.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

None

Sublethal observations / clinical signs:

Test Conditions and Water Quality Measurements

Table 6.1.1/1: Summary of physico-chemical parameters in the test solutions.

Parameter	Minimum	Maximum	Range	Number
pH	7.5	7.6	0.1	12
Oxygen [mg/L]	8.5	8.9	0.4	12
Oxygen [% of air saturation]	101	104	3	12
Temperature [°C]	22.5	23.5	1.0	12
Temperature^[a][°C]	21.9	23.2	1.3	199

^[a]automatic measurement of the control test vessel.

 

Biological Results

There was no mortality, either in the control nor in the test item treatment; limit concentration at 3.00 mg test item/L, during the 96-hour exposure period.

 

Results Related to the Behaviour of Fish

There were no behavioural differences in fish in the treated concentrations compared with the control fish.

 

Analytically Determined Test Item Concentrations (Summary)

Table 6.1.1/2: Summary of the analytical results at the beginning and at the end of the test.

Test period [h]	Age of test solution [h]	Treatment code	Nominal concentration [mg/L]	Measured concentration; expressed as test item [mg/L]	Recovery in % of nominal concentration [%]
0	0	C0 (control)	0	<LOD	–
0	0	C1 (fresh)	3.00	2.56	85
48	48	C1 (aged)	3.00	2.31	77
48	0	C1 (fresh)	3.00	2.63	88
96	48	C0 (control)	0	<LOD	–
96	48	C1 (aged)	3.00	2.31	77

 

The concentration measured in the limit concentration level in the freshly prepared solution at start of the exposure and after 48 hours were 2.56 and 2.63 mg test item/L, equivalent to recoveries of 85 and 88% of the nominal concentration and slightly decrease during the exposure time of 48 hours (48 and 96 hours: 2.31 mg test item/L with 77% of nominal). Since not all the measured concentrations were within ±20% of the nominal concentration, the biological data were evaluated based on nominal and mean measured concentrations as mg test item/L.

The geometric mean measured concentration throughout the exposure period was calculated to be 2.45 mg test item/L.

Validity criteria fulfilled:

yes

Conclusions:

In a GLP study on the acute toxicity to Zebrafish (Danio rerio) according to OECD203 "Fish, Acute Toxicity Test", The concentrations measured in the limit concentration level at the start of each renewal period were 2.56 (0 hours) and 2.63 (48 hours) mg test item/L and slightly decreased during the exposure period (48 and 96 hours: 2.31 mg test item/L).
Since not all of the measured concentrations were within ±20% of the nominal concentration, the biological data were evaluated based on the nominal limit concentration and the mean measured concentration, expressed as mg test item/L.
No mortalities occurred during the 96 hours exposure of the fish at 3.00 mg test item/L. Therefore, it can be concluded that the LC50 (96 h) to fish (Danio rerio) is:
- higher than 3.00 mg test item/L based on the nominal concentration and
- higher than 2.45 mg test item/L based on the mean measured concentration.

Executive summary:

In a GLP study on the acute toxicity to Zebrafish (Danio rerio) according to OECD203 "Fish, Acute Toxicity Test", a limit concentration / threshold concentration of the test item in aqueous solution was prepared. The test animals were exposed to this concentration as well as to a control without the test item for a test period of 96 hours. During the test period the test solutions were renewed once (semi-static system). Reconstituted water according to OECD 203 was used as a test medium.

All fish in the test were observed 2, 5, 19, 24, 48, 49, 52, 67, 72, 91 and 96 hours after start of exposure for any effects other than lethality: effects on the appearance, size and behaviour of the fish that make them clearly distinguishable from the control animals, e.g., different swimming behaviour, changes in appearance of the fish were recorded.

Test solutions were analysed for the concentration of the test item. The concentrations measured in the limit concentration level at the start of each renewal period were 2.56 (0 hours) and 2.63 (48 hours) mg test item/L and slightly decreased during the exposure period (48 and 96 hours: 2.31 mg test item/L).

No mortalities occurred during the 96 hours exposure of the fish at 3.00 mg test item/L. Therefore, it can be concluded that the LC₅₀(96 h) to fish (Danio rerio) is higher than 3.00 mg test item/L based on the nominal concentration and higher than 2.45 mg test item/L based on the mean measured concentration.

All validity criteria were fulfilled as required by the study plan.

Description of key information

Two  toxicity tests were conducted on the registered substance to address short-term toxicity to fish.

For animal welfare reasons, an in vitro assay has been performed and a limit test according to OECD 203.

The objective of the in vitro study (Laue, 2020) was to determine the effects of Ethyl Safranate on the viability of the RTgill-W1 cell line (from rainbow trout) to predict the fish acute toxicity. The in vitro EC50 value for Ethyl Safranate was 27.06 mg/L based on mean measured concentrations, and the predicted acute LC50 value for fish was 10.7 mg/L.

In the in vivo study (Gilberg, 2020), the acute toxicity of the test item to Danio rerio  was determined after 96 hour following the OECD 203 guideline under GLP conditions. A limit concentration of 3mg/L (nominal) was chosen based on the lowest EC50-value of existing and reliable algal and acute daphnia toxicity (e.g. algae ErC50 of 3.89mg/L). No mortalities occurred during the 96 hours exposure of the fish at 3.00 mgL based on the nominal concentration; 2.45 mg/L based on mean measured concentrations. 

From the in vivo study it can only be concluded that the LC50 is higher than the limit concentration and, given that this was chosen based on the algae ErC50, indicates that algae is the more senstive species. For the purpose of classification and labelling an "actual" LC50 is preferred. Therefore the predicted LC50 of 10.7mg/L has been chosen as the key value for the fish endpoint. PNEC derivation will be based on the most sensitive species, e.g. the algae ErC50 of 3.89mg/L. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Remarks:

Based on measured concentrations.

Effect concentration:

10.7 mg/L

Additional information

Acute Toxicity (OECD 203):

The acute toxicity of the test item Ethyl Safranate to juvenile fish of the species zebrafish (Danio rerio) was determined in a 96-hour test according to the OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 2019. A limit test at the nominal test item concentration of 3 mg/L was performed.

The concentration measured in the limit concentration level at the start of each renewal period were 2.56 (0 hours) and 2.63 (48 hours) mg test item/L and slightly decreased during the exposure period (48 and 96 hours: 2.31 mg test item/L).

Since the measured concentrations at the end of the exposure period were not within ±20% of the nominal concentrations, the biological data were evaluated based on the nominal limit concentration and the mean measured concentration, expressed as mg test item/L.

No mortalities occurred during the 96 hours exposure of the fish at 3.00 mg test item/L. Therefore, it can be concluded that the LC50 (96 h) to fish (Danio rerio) is:

- higher than 3.00 mg test item/L based on the nominal concentration and

- higher than 2.45 mg test item/L based on the mean measured concentration.

 

RTgill-W1 assay:

Acute toxicity to fish is traditionally tested according to OECD technical guideline (TG) 203 which requires the use of animals and monitoring mortality. Since in fish the gills are the organs which are directly exposed to dissolved organic chemicals, acute toxicity is often related to toxicity to the gills. A straightforward in vitro assay was thus developed based on cytotoxicity to a static monolayer culture of a permanent rainbow trout gill cell line (RTgill-W1) [1]. An international ring-trial showed this assay to be transferable and to produce highly reliable and reproducible results [2]. An ISO standard has been published to determine water quality (e.g. for effluent testing) using the RTgill-W1 assay [3]. The RTgill-W1 assay completely replaces the use of vertebrates and has recently been adopted as a new OECD guideline; TG No. 249 Fish Cell Line Acute Toxicity: The RTgill-W1 cell line assay (adopted 14 June, 2021).

In terms of its applicability to fragrance chemicals, a benchmarking study has been performed covering a broad range of physico-chemical properties and diverse chemistries, and, possessing good quality in vivo fish toxicity studies [4]. A strong correlation (R2 = 0.90–0.94) between the logarithmic in vivo LC50 values, based on fish mortality, and the logarithmic in vitro median effect concentration (EC50) values based on cell viability was observed.

The effects of Ethyl Safranate on the viability of the RTgill-W1 cell line were assessed to predict the fish acute toxicity. The RTgill-W1 cells were seeded in 24-well plates and exposed in minimal medium to six different test concentrations and controls without chemical in triplicate for 24 h at 19°C. GC-analysis of the test chemical concentrations in media was conducted at the start (0 h) and the end (24 h) of exposure. Cell viability was determined at 24 h using metabolic activity as endpoint (PrestoBlue® assay). The mean measured test chemical concentrations were 57.68, 47.19, 29.69, 15.60, 7.44 and 3.83 mg/L (nominal concentrations 256, 128, 64, 32, 16, 8 mg/L). The EC50 was determined to be 27.06mg/L based on mean measured concentrations.  In order to predict a fish in vivo LC50 value from the in vitro EC50, the following regression equation from the study of Natsch et al. [4] was used: Log LC50 (fish in vivo) = (0.97 × log EC50 PB mean measured) - 0.36; n = 37, R2 = 0.94. The predicted LC50 value for fish was 10.7 mg/L.

 

REFERENCES

[1] 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.

[2] 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.

[3] ISO, Water quality — Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1), in ISO 21115:2019. 2019.

[4] 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.