2,4-dimethyl-4-phenyltetrahydrofuran

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:

supporting study

Study period:

22 May and 26 June 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test guideline is currently under evaluation

Qualifier:

according to guideline

Guideline:

other: Project 2.63: New TG on Fish cell line acute toxicity test - RTgill-W1 cell line assay

Version / remarks:

currently under evaluation by OECD

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

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations: 128, 64, 32, 16, 8, 4 mg/L
- Sampling method: 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.

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Serial half dilutions in DMSO
- Controls: DMSO
- Chemical name of vehicle: Dimethylsulfoxide
- Concentration of vehicle in test medium: 0.5%

Test organisms (species):

other: The rainbow trout (Oncorhynchus mykiss) RTgill-W1 cell line

Details on test organisms:

TEST ORGANISM
- Common name: The rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Swiss center for aquatic research EAWAG (Dübendorf, Switzerland)

Test type:

other: in vitro

Total exposure duration:

24 h

Test temperature:

19°C

Nominal and measured concentrations:

Nominal concentrations: 128, 64, 32, 16, 8, 4 mg/L
Mean measured concentrations: 112.1, 50.1, 21.4, 6.8, 3.4, 1.7 mg/L

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline (DCA)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

68.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Cytotoxicity

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

26.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Predicted in vivo acute toxicity (LC50)

Sublethal observations / clinical signs:

Chemical Analysis

Test substance was quantified based on the sum of the peak area of the two major peaks (Appendix A and B). Representative chromatograms of standard solutions and a typical calibration curve are presented in Appendix A. Representative chromatograms of test samples are presented in Appendix B. Calibrations were prepared based on the nominal concentrations used in the cell culture plates (4 - 128 mg/L). The limit of quantification (LOQ) for Test substance in medium using this method was below the lowest calibration standard (4 mg/L). Since the measured concentrations of Test substance in the two lowest exposure levels (nominal concentrations: 4 and 8 mg/L) declined below the LOQ, the mean measured concentrations for were derived from the lowest mean measured concentrations (6.8 mg/L; nominal: 16 mg/mL). However, this extrapolation does not affect the results, since the EC50 based on mean measured concentration was > 8 mg/mL.

 

Measured concentrations of the test samples ranged from below LOQ to 160.9 mg/L at T0h and from below LOQ to 63.3 mg/L at T24h.

 

Table 6.1.1/1: Measured exposure concentrations of test substance

Nominal concentrations (mg/L)	Measured concentrations (mg/L)
	0 h	24 h	Mean
128	160.9	63.3	112.1
64	72.8	27.4	50.1
32	32.8	9.9	21.4
16	12.4	1.3	6.8
8	2.6	< LOQ	3.4*
4	< LOQ	< LOQ	1.7*

*These concentrations were derived from the lowest mean measured concentration (6.8 mg/L).

 

Cytotoxicity test

The three acceptance criteria were fulfilled except (i) The DMSO versus solvent-free control wells did not differ in raw fluorescence values by more than 10 % (5.5 % 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 (2.2 % 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 four lowest test concentrations (nominal concentrations 4 - 32 mg/L) at 24 h. The NOEC was thus considered to be 21.4 mg/L (nominal concentration 32 mg/L). The EC50 value based on mean measured concentration was 68.77 mg/L. Based on the regression equation, an in vivo LC50 of 26.44 mg/L is predicted.

 

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

Nominal conc.	128 mg/L	64 mg/L	32 mg/L	16 mg/L	8 mg/L	4 mg/L
Mean measured conc.	112.1 mg/L	50.1 mg/L	21.4 mg/L	6.8 mg/L	3.4 mg/L	1.7 mg/L
Rep 1 (% viability)	6.3	86.5	96.6	97.9	92.6	92.6
Rep 2 (% viability)	5.3	87.8	99.7	102.4	103.9	91.1
Rep 3 (% viability)	6.7	82.0	94.2	97.5	94.7	96.7
Average (% viability)	6.1	85.5	96.8	99.3	97.0	93.5
Standard deviation	0.8	3.0	2.8	2.7	6.0	2.9
Coefficient of variation (%)	12.4	3.6	2.8	2.8	6.2	3.1

  

Table 6.1.1/3: Effect concentrations, LOEC and NOEC of test substance in the RTgill-W1 cell line.

Effect concentrations expressed as mean measured concentration (mg/L)
	Cytotoxicity 24 h (mg/L)	Predicted in vivo acute toxicity (LC50) (mg/L)
EC50	68.77 (63.34 – 74.66)^1mg/L	26.44
LOEC	50.1 mg/L	n.a.^2
NOEC	21.4 mg/L	n.a.

1 Values in brackets are 95% confidence limits

2 n.a. not applicable

 

The following equation is derived (based on EC₅₀mean measured) to predict the in vivo outcome:

 

Log LC50_{fish in vivo}= (0.97 × log EC50 PrestoBlue®_{gill mean measured}) – 0.36

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value based on mean measured concentrations in the RTgill-W1 cell assay was 68.77 mg/L. Based on a regression equation developed with 37 fragrance molecules an in vivo LC50 of 26.44 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 4 – 128 mg/L based on nominal concentrations (measured concentrations ranging from below the limit of quantification (LOQ) – 160.9 mg/L at the start and below LOQ – 63.3 mg/L at the end of the exposure).

The mean measured test chemical concentrations were 112.1, 50.1, 21.4, 6.8, 3.4, 1.7 mg/L (nominal concentrations 128, 64, 32, 16, 8, 4 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	68.77 (63.34 – 74.66)^1mg/L	26.44
LOEC	50.1 mg/L	n.a.^2
NOEC	21.4 mg/L	n.a.

1 Values in brackets are 95% confidence limits

2 n.a. not applicable

 

The concentrations at which no significant (≤ 10%) dose related cytotoxicity occurred were ≤ 21.4 mg/L (nominal 32 mg/L). The NOEC was thus considered to be 21.4 mg/L. After 24 hours, the EC₅₀ value based on mean measured concentration was 68.77 mg/L. Based on the regression equation derived from a training set of 37 fragrance molecule, an in vivo LC₅₀ of 26.44 mg/L is predicted.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August to 10 October 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 (incl. QA statement)

Remarks:

Date of Inspection: 21 to 30 August 2017 / Date of Decision: 10 January 2018

Specific details on test material used for the study:

Storage: dry, well ventilated, preferably full, hermetically sealed
Temperature: Ambient / 10-30°C (50-85°F)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 20.0 mg/L

- Sampling method: For measurement of the actual concentration of the test item in this semi-static test with daily test medium renewal, duplicate 10 mL samples were taken from the test medium and the control in small glass vials at the start and the end of each test medium renewal period.

The concentrations of the test item in the test medium and the control were analytically measured in one of the duplicate samples from all sampling times. The other duplicate sample remained as back-up stored frozen.

- Sample storage conditions before analysis: Each sample was stored frozen (at –20 ± 5 °C) and protected from light immediately after sampling until analysis.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio
- Source: A breeding culture at IES Laboratories and were from the same batch
- Length at study initiation (length definition, mean, range and SD): 1.8 ± 0.07 cm (1.7 to 1.9)
- Weight at study initiation (mean and range, SD): 0.05 ± 0.01 g

ACCLIMATION
- Acclimation period: At least one week
- Acclimation conditions: Same as test conditions
- Type and amount of food during acclimation: commercial fish diet (TetraMin® Hauptfutter, supplied by TETRA-Werke, 49324 Melle / Germany)
- Feeding frequency during acclimation: at least six days the week
- Health during acclimation: No mortality observed

FEEDING DURING TEST: Not fed

Test type:

semi-static

Water media type:

other: Reconstituted test water

Limit test:

yes

Total exposure duration:

96 h

Hardness:

125 mg/L (as CaCO3)

Test temperature:

21 °C

pH:

7.4

Dissolved oxygen:

8.4 to 8.5 mg/L at the start of the test medium renewal periods and slightly decreased to 7.6 – 7.8 mg/L in the closed system during the 24-hour renewal periods.

Nominal and measured concentrations:

nominal concentration: 20 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass bottle
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 5 L, without head space
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): 24 h

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Ca/mg ratio: 4:1

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour dark
- Light intensity: 10.0 and 10.6 μE s-1 m-2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormalities (twice every 24-hour)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Deviation of measured concentrations from nominal concentrations was smaller than 20% throughout the test. Therefore, according to the OECD 203 guideline, the result can be based on nominal concentrations.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 17.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Analytical Results

The measured concentrations of the test item in the test medium at the start and end of the four renewal periods are shown in the table below.

Table 6.1.1/1: Results for Test Samples

Sampling Day/ Sample Age (day/hours)	Nominal Concentration of Test Item Cnom(mg/L)	Measured Concentration of Test Item x (mg/L)	Sample Preparation Factor F	Determined Concentration of Test Item c (mg/L)	% of Nominal (%)
0/0 (fresh)	Control 20	n.d. 45.9	0.4 0.4	<LOQ 18.4	n.a. 92
1/24 (aged)	Control 20	n.d. 41.8	0.4 0.4	<LOQ 16.7	n.a. 84
1/0 (fresh)	Contro l20	n.d. 43.3	0.4 0.4	<LOQ 17.3	n.a. 87
2/24 (aged)	Control 20	n.d. 46.4	0.4 0.4	<LOQ 18.6	n.a. 93
2/0 (fresh)	Control 20	n.d. 43.4	0.4 0.4	<LOQ 17.4	n.a. 87
3/24 (aged)	Control 20	n.d. 44.6	0.4 0.4	<LOQ 17.8	n.a. 89
3/0 (fresh)	Control 20	n.d. 44.4	0.4 0.4	<LOQ 17.8	n.a. 89
4/24 (aged)	Control 20	n.d. 44.1	0.4 0.4	<LOQ 17.7	n.a. 88

LOQ: 2.08 mg test item / L

n.d. = not detected

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

The measured concentrations of test item in the freshly prepared test media at the start of the four test medium renewal periods were between 87 and 92 % of the nominal value of 20.0 mg/L, demonstrating the correct preparation of the test media.

The measured test item concentrations in the aged test media at the end of the test medium renewal periods of 24 hours were between 84 and 93 % of the nominal concentration.

Therefore, by applying a semi-static test design the exposure concentration could be maintained as constant as possible during the total exposure period of 96 hours. In consequence, the test results are based on nominal concentration of 20.0 mg/L.

In addition, for a more conservative interpretation, the biological results are also given based on the mean measured concentration of 17.7 mg/L (calculated as the arithmetic mean of the four geometric means determined for the four 24-hour test medium renewal periods).

Table 6.1.1/2: The measured concentrations of test item

Renewal Period (24 h each)	Analytically Measured Concentrations of the Test Item*				Mean Measured Concentration for each Renewal Period (geometric mean) (mg/L)	Mean Measured Concentration for the Total Test Period (arithmetic mean) (mg/L)
	at the Start of the Renewal Period		at the End of the Renewal Period
	(mg/L)	(% of nominal)	(mg/L)	(% of nominal)
1	18.4	92	16.7	84	17.5	17.7
2	17.3	87	18.6	93	17.9
3	17.4	87	17.8	89	17.6
4	17.8	89	17.7	88	17.8

*: in test medium prepared at a nominal test item concentration of 20.0 mg/L.

Biological Results

In the control and the sole tested test item concentration all fish survived until the end of the test and no visible abnormalities were observed at the test fish.

Table 6.1.1/3: Cumulative Mortality and Visible Abnormalities Observed in the Test Fish

Treatment [mg/L]		Cumulative Mortality and Visible Abnormalities [Number of Fish]
		Observation Time
		Day 0		Day 1		Day 2		Day 3		Day4
		p.m. (2 hours after start)	p.m. (5 hours after start)	a.m.	p.m.	a.m.	p.m.	a.m.	p.m.	a.m.	p.m.
Control	M	0	0	0	0	0	0	0	0	0	0
	VA	0	0	0	0	0	0	0	0	0	0
20.0* (17.7) **	M	0	0	0	0	0	0	0	0	0	0
	VA	0	0	0	0	0	0	0	0	0	0
LC50***		> 20.0 (> 17.7)		> 20.0 (> 17.7)		> 20.0 (> 17.7)		> 20.0 (> 17.7)		> 20.0 (> 17.7)

M: Cumulative mortality (Cumulative number of dead fish, 7 fish were inserted).

VA: Visible abnormalities (Number of fish showing visible abnormalities).

a.m./p.m.: Readings in the morning / afternoon of the respective observation day.

*: 20.0 mg/L was the nominal test item concentration of test item.

**: 17.7 mg/L was the mean measured concentration of test item during the 96-h test period in test medium, which is given here for a more conservative assessment.

***: LC50 in mg/L, based on the nominal and (in brackets) the mean measured test item concentration and the readings at 2, 5, 24, 48, 72 and 96 hours after test start (i.e. the insertion of the test organisms).

 

Therefore, the 96-hour NOEC and LC₀ of test item to zebra fish were both determined to be at least 20.0 mg/L (nominal concentration) or 17.7 mg/L (mean measured concentration). The 96-hour LC₅₀ and LC₁₀₀ could not be quantified due to the absence of toxicity of test item at the tested concentration and were clearly higher than 20.0 mg/L (nominal concentration) or 17.7 mg/L (mean measured concentration).

Table 6.1.1/4: The biological results

Endpoints	Concentration oftest item
	based on the nominal test item concentration	based on the mean measured concentration
96-hour LC50	> 20.0 mg/L	> 17.7 mg/L
96-hour LC0	≥ 20.0 mg/L	≥ 17.7 mg/L
96-hour LC100	> 20.0 mg/L	> 17.7 mg/L
96-hour NOEC	≥ 20.0 mg/L	≥ 17.7 mg/L
96-hour LOEC	> 20.0 mg/L	>17.7 mg/L

 

In conclusion, the test demonstrated that the test organism fish is not the most sensitive test organism compared to daphnia and algae. According to Sponsor information the E_rC₅₀ value determined in an algae study (OECD 201) was 24 mg/L and the EC₅₀ value determined in an acute daphnia study (OECD 202) was 18 mg/L.

 

Results of Monitoring of Experimental Conditions

No remarkable observations were made concerning the appearance of the test media. They were clear solutions throughout the test media renewal periods of 24 hours.

The pH values measured in the test medium and the control were constantly at 7.4 during the test period. The dissolved oxygen concentration was in the range of 8.4 to 8.5 mg/L at the start of the test medium renewal periods and slightly decreased to 7.6 – 7.8 mg/L (i.e. 85 – 87 % saturation) in the closed system during the 24-hour renewal periods. Therefore, the oxygen concentration never dropped below 60 % of the air saturation value. The water temperature was measured to be 21 °C during the test period.

Table 6.1.1/5: pH Values in the Freshly Prepared and Aged Test Medium and the Control

Treatment [mg/L]	pH Value
	Exposure Time [Hours]
	0 - 24		24 - 48		48 - 72		72 - 96
	fresh	aged	fresh	aged	fresh	aged	fresh	aged
Control	7.4	7.4	7.4	7.4	7.4	7.4	7.4	7.4
20.0*	7.4	7.4	7.4	7.4	7.4	7.4	7.4	7.4

*: Nominal test item concentration of test item.

 

Table 6.1.1/6: Oxygen Concentrations [mg/L] in the Freshly Prepared and Aged Test Medium and the Control

Treatment [mg/L]	Oxygen Concentration [mg/L]
	Exposure Time [Hours]
	0 - 24		24 - 48		48 - 72		72 - 96
	fresh	aged	fresh	aged	fresh	aged	fresh	aged
Control	8.5	7.8	8.5	7.7	8.5	7.7	8.4	7.6
20.0*	8.5	7.8	8.5	7.7	8.5	7.7	8.4	7.6

*: Nominal test item concentration of test item.

 

Table 6.1.1/87: Temperatures [°C] in the Freshly Prepared and Aged Test Medium and the Control

Treatment [mg/L]	Water Temperature [°C]
	Exposure Time [Hours]
	0 - 24		24 - 48		48 - 72		72 - 96
	fresh	aged	fresh	aged	fresh	aged	fresh	aged
Control	21	21	21	21	21	21	21	21
20.0*	21	21	21	21	21	21	21	21

*: Nominal test item concentration of test item.

Validity criteria fulfilled:

yes

Conclusions:

The test item had no acute toxic effects on juvenile Zebrafish (Danio rerio) at the sole tested concentration of 20.0 mg/L (nominal concentration) or 17.7 mg/L (mean measured concentration) under the tested conditions (i.e. semi-static test with 24 h renewal periods in a closed system). Therefore, the 96-hour LC50 for fish was determined to be > 20.0 mg/L (nominal concentration).

According to the threshold approach, it was demonstrated that the test organism fish is not the most sensitive species for the test item, since this concentration was higher than the threshold concentration of 18 mg/L, i.e. the lower EC50 value determined in an algae study (OECD 201) and an acute daphnia study (OECD 202).

This valid test was performed according to the OECD Guideline No. 203, Fish, Acute Toxicity Test, 2019.

Executive summary:

The acute toxicity of the test item to juvenile fish of the species Zebrafish (Danio rerio) was determined in a 96-hour test according to the OECD Guideline No. 203, Fish, Acute Toxicity Test, 2019.

 

According to the guideline, a limit test with only one test concentration was performed to demonstrate that the test organism fish is not the most sensitive test organism to test item compared to algae and daphnia. The test method is based on the threshold approach as developed for chemical substances at the European Commission’s Joint Research Centre.

 

Zebra fish (Danio rerio) were exposed to an aqueous test medium containing the test item at the threshold concentration of nominal 20.0 mg/L. A control (test water without test item) was tested in parallel. The selected test concentration of 20.0 mg/L corresponded approximately to the lower EC₅₀value determined in an algae study (OECD 201) and an acute daphnia study (OECD 202).

 

The test was performed in closed glass vessels (closed system without head space) completely filled with test medium, to avoid potential losses of test item by evaporation. A semi-static test design with daily water renewal was chosen to keep the concentration of the test item as constant as possible during the exposure period of 96 hours and to provide sufficient oxygen saturation in the closed system.

 

The test design was based on the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.

 

In the test medium samples from the start of the 24-hour test medium renewal periods the measured test item concentrations ranged between 87 and 92 % of the nominal value of 20.0 mg/L, demonstrating the correct dosage of the test item.

 

The measured test item concentrations in the aged test media at the end of the test medium renewal periods of 24 hours were between 84 and 93 % of the nominal concentration. Therefore, by applying a semi-static test design the exposure concentration could be maintained as constant as possible during the total exposure period of 96 hours. In consequence, the test results are based on nominal concentration of 20.0 mg/L.

 

In addition, for a more conservative interpretation, the biological results are also given based on the mean measured concentration of 17.7 mg/L (calculated as the arithmetic mean of the four geometric means determined for the four 24-hour test medium renewal periods).

Renewal Period (24 h each)	Analytically Measured Concentrations of the Test Item*				Mean Measured Concentration for each Renewal Period (geometric mean) (mg/L)	Mean Measured Concentration for the Total Test Period (arithmetic mean) (mg/L)
	at the Start of the Renewal Period		at the End of the Renewal Period
	(mg/L)	(% of nominal)	(mg/L)	(% of nominal)
1	18.4	92	16.7	84	17.5	17.7
2	17.3	87	18.6	93	17.9
3	17.4	87	17.8	89	17.6
4	17.8	89	17.7	88	17.8

*: in test medium prepared at a nominal test item concentration of 20.0 mg/L.

 

At the start of the test, seven fish were introduced into each test vessel in a random order. Every 24 hours, the surviving fish were carefully transferred into a new test vessel with freshly prepared test medium. The fish were observed for visible abnormalities and mortality twice every 24-hour period.

 

All fish in the test survived until the end of the 96 hours exposure period and no toxic effect on the test organism fish were observed.

 

The biological results after 96 hours of exposure to test item, related to the nominal and the mean measured test item concentration in the test medium were as presented in the table below:

Endpoints	Concentration oftest item
	based on the nominal test item concentration	based on the mean measured concentration
96-hour LC50	> 20.0 mg/L	> 17.7 mg/L
96-hour LC0	≥ 20.0 mg/L	≥ 17.7 mg/L
96-hour LC100	> 20.0 mg/L	> 17.7 mg/L
96-hour NOEC	≥ 20.0 mg/L	≥ 17.7 mg/L
96-hour LOEC	> 20.0 mg/L	>17.7 mg/L

 

In conclusion, the test demonstrated that the test organism fish is not the most sensitive test organism compared to daphnia and algae.

 

Validity Criteria:

The test was considered valid as no fish died in the control and the tested threshold concentration during the 96-hour test period. The oxygen concentration in the test medium and the control did not drop below 60 % of the air saturation during the test.

Description of key information

OECD Guideline 203, GLP, key study, validity 1:

96h-LC50 (Danio rerio) > 20.0 mg/L (nominal); Deviation of measured concentrations from nominal concentrations was smaller than 20% throughout the test. Therefore, according to the OECD 203 guideline, the result can be based on nominal concentrations.

96h-LC50 (Danio rerio) > 17.7 mg/L (measured, geometric mean);

Additional information

To assess the acute toxicity of the substance to fish, two studies are available:

• The key acute toxicity study was performed on tested susbtance to Zebrafish (Danio rerio) according to the OECD 203 with GLP statement.
  

Zebra fish (Danio rerio) were exposed to an aqueous test medium containing the test item at the threshold concentration of nominal 20.0 mg/L. A control (test water without test item) was tested in parallel. The selected test concentration of 20.0 mg/L corresponded approximately to the lower EC₅₀value determined in an algae study (OECD 201) and an acute daphnia study (OECD 202).

The test was performed in closed glass vessels (closed system without head space) completely filled with test medium, to avoid potential losses of test item by evaporation. A semi-static test design with daily water renewal was chosen to keep the concentration of the test item as constant as possible during the exposure period of 96 hours and to provide sufficient oxygen saturation in the closed system.

In the test medium samples from the start of the 24-hour test medium renewal periods the measured test item concentrations ranged between 87 and 92 % of the nominal value of 20.0 mg/L, demonstrating the correct dosage of the test item.

The measured test item concentrations in the aged test media at the end of the test medium renewal periods of 24 hours were between 84 and 93 % of the nominal concentration. Therefore, by applying a semi-static test design the exposure concentration could be maintained as constant as possible during the total exposure period of 96 hours. In consequence, the test results are based on nominal concentration of 20.0 mg/L.

In addition, for a more conservative interpretation, the biological results are also given based on the mean measured concentration of 17.7 mg/L (calculated as the arithmetic mean of the four geometric means determined for the four 24-hour test medium renewal periods).

 

The biological results after 96 hours of exposure to test item, related to the nominal and the mean measured test item concentration in the test medium were as presented in the table below:

Endpoints	Concentration of test item
	based on the nominal test item concentration	based on the mean measured concentration
96-hour LC50	> 20.0 mg/L	> 17.7 mg/L

 

In conclusion, the test demonstrated that the test organism fish is not the most sensitive test organism compared to daphnia and algae.

 

• The supporting 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 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 4 – 128 mg/L based on nominal concentrations (measured concentrations ranging from below the limit of quantification (LOQ) – 160.9 mg/L at the start and below LOQ – 63.3 mg/L at the end of the exposure).

 

The mean measured test chemical concentrations were 112.1, 50.1, 21.4, 6.8, 3.4, 1.7 mg/L (nominal concentrations 128, 64, 32, 16, 8, 4 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.

 

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	68.77 (63.34 – 74.66)^1mg/L	26.44

1 Values in brackets are 95% confidence limits

Based on the regression equation derived from a training set of 37 fragrance molecule, an in vivo LC₅₀ of 26.44 mg/L is predicted.