Reaction mass of 3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate and 3,7-dimethyloctyl 3-methyl-2-butenoate and citronellyl 3-methylcrotonate

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:

from 22.05.2019 to 28.10.2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The method has been validated and published by ISO (ISO21115) and is currently under evaluation as a new test guideline by the OECD.

Justification for type of information:

SINODOR CQ is used exclusively 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.

The non-animal alternative method described herein, has been validated and published by the International Organization for Satndardization (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, is currently at an advanced stage of evaluation in the OECD Test Guideline Programme. 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.

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.

Version / remarks:

April, 2019

Deviations:

no

Principles of method if other than guideline:

SINODOR CQ is used exclusively 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.

The non-animal alternative method described herein, has been validated and published by the International Organization for Satndardization (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, is currently at an advanced stage of evaluation in the OECD Test Guideline Programme. 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.

The method is currently under evaluation as a new test guideline by the OECD.
The study described in this report has been 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 International
Organization for Standardization test method ISO 21115:2019.
This in vitro method was demonstrated to have an excellent performance to predict acute toxicity of chemicals with a narcotic mode of action. A benchmarking study on 38 fragrance chemicals with known fish acute toxicity was performed recently and the resulting regression equation was used to predict the acute fish toxicity for Sinodor CQ. The 38 fragrance chemicals covered a wide range of physicochemical properties and lethal concentration (LC50) values. The RTgill-W1 cells are exposed to six different test chemical concentrations and cell viability is determined after 24 h of exposure and the test chemical concentrations are analysed at the beginning and end of the 24 h exposure.

GLP compliance:

no

Remarks:

Testing done in internal lab

Specific details on test material used for the study:

Batch number: VE00571754
Expiry date: 18 March, 2020
Purity: 99.7% (sum of isomers)
Storage conditions: Ambient temperature in dark, dry conditions

Analytical monitoring:

yes

Details on sampling:

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.

Vehicle:

yes

Remarks:

Dimethylsulfoxide (DMSO) at 0.5% in Liebovitz (L15) medium

Details on test solutions:

Test Plate Dosing and Incubation
For the first experiment, 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).
For the second experiment, the test chemical was dissolved to a final concentration of 6.4 mg/mL in DMSO. Serial dilutions in DMSO were prepared to a final concentration of 6.4, 3.2, 1.6, 0.8, 0.4 and 0.2 mg/mL. From each DMSO solution, a dosing solution was prepared by adding 50 µL to 9.95 mL of exposure medium L15/ex (final nominal test chemical concentrations: 32, 16, 8, 4, 2, 1 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 2.5 mL dosing solution was added to each well. Solvent controls received 2.5 mL L15/ex containing 0.5% DMSO. Directly after addition of dosing solution, 0.5 mL of exposure solution was sampled from each well for chemical analysis. The plates, now containing 2 mL exposure solution in each well, were 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:

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: Liebovitz L15 medium

Limit test:

no

Total exposure duration:

24 h

Test temperature:

19°C

Nominal and measured concentrations:

Two experiments were performed. The first experiment was conducted at 128, 64, 32, 16, 8, 4 mg/L based on nominal concentrations (11.85, 17.58, 11.43, 7.90, 4.28, 2.01 mg/L based on mean measured concentrations). The second test was conducted at 32, 16, 8, 4, 2, 1 mg/L based on nominal concentrations (8.41, 6.03, 3.04, 1.70, 0.97, 0.68 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 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:

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.

Reference substance (positive control):

yes

Remarks:

3,4 dichloroaniline (DCA)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.53 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cytotoxicity

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

1.89 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cytotoxicity

Remarks on result:

other: predicted in vivo acute toxicity

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

0.97 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks on result:

other: Cytotoxicity

Details on results:

The three acceptance criteria discussed by Fischer et al. (2019) were fulfilled: (i) The DMSO versus solvent-free control wells did not differ in raw fluorescence values by more than 10% reduction: 9.9 and 5.2 % reduction in control without solvent measured in experiment 1 and 2, respectively; (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 (3.5 and 0.1% variation measured in experiment 1 and 2, respectively); (iii) EC50 values of the positive control (DCA) were within the given range based on the international validation study as discussed below.
In the first experiment, 83% cell viability (average) was observed for the lowest test concentration (nominal concentration 4 mg/L, mean measured 2.01 mg/L) at 24 h. The concentration at which no significant (≤ 10%) dose related cytotoxicity occurred was < 2.01 mg/L (i.e. the lowest mean measured concentration). Therefore, the NOEC was considered to be < 2.01 mg/L. The EC50 value based on mean measured concentration was 4.76 mg/L. Based on a regression equation derived from a training set of 37 fragrance molécules, an in vivo LC50 of 1.98 mg/L is predicted.

No significant (≤ 10%) dose related cytotoxicity occurred was observed at the two lowest test concentrations (nominal concentrations 1 and 2 mg/L) at 24 h in the second experiment. The NOEC was thus considered to be 0.97 mg/L (nominal concentration 2 mg/L). The EC50 value based on mean measured concentration was 4.29 mg/L. Based on the regression equation, an in vivo LC50 of 1.79 mg/L is predicted.

Results with reference substance (positive control):

EC50 values of the positive control (DCA) were within the given range based on the international validation study.
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:

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 et al., 2018). This led to a predictive regression equation based on the EC50 determined with PrestoBlue® (PB):
Log LC50fish in vivo = (0.97 × log EC50PB mean measured) - 0.36
This regression equation was used to predict an in vivo LC50.

Sublethal observations / clinical signs:

Detailed cell viability data (% viability as compared to solvent control using the PrestoBlue® assay) - first experiment.

Nominal conc.

128 mg/L

64 mg/L

32 mg/L

16 mg/L

8 mg/L

4 mg/L

Mean measured conc.

11.9 mg/L

17.6 mg/L

11.4 mg/L

7.9 mg/L

4.3 mg/L

2.0 mg/L

Rep 1 (% viability)

19.8

6.0

20.2

29.8

48.7

83.3

Rep 2 (% viability)

16.5

13.5

38.0

49.6

35.7

81.6

Rep 3 (% viability)

24.4

19.8

32.7

33.6

43.6

83.9

Average (% viability)

20.2

13.1

30.3

37.7

42.7

83.0

Standard deviation

4.0

6.9

9.1

10.5

6.6

1.2

Coefficient of variation (%)

19.6

52.8

30.0

27.9

15.4

1.4

 

 

 

Detailed cell viability data (% viability as compared to solvent control using the PrestoBlue® assay) - second experiment.

Nominal conc.

32 mg/L

16 mg/L

8 mg/L

4 mg/L

2 mg/L

1 mg/L

Mean measured conc.

8.41 mg/L

6.03 mg/L

3.04 mg/L

1.70 mg/L

0.97 mg/L

0.68 mg/L

Rep 1(% viability)

9.8

42.7

96.1

100.4

102.5

107.6

Rep 2 (% viability)

9.6

27.7

83.0

98.7

102.7

110.6

Rep 3 (% viability)

9.1

12.0

49.2

66.8

78.8

91.1

Average (% viability)

9.5

27.5

76.1

88.6

94.6

103.1

Standard deviation

0.3

15.4

24.2

18.9

13.8

10.5

Coefficient of variation (%)

3.5

56.0

31.8

21.4

14.5

10.2

 

 

Effect concentrations, LOEC and NOEC of Sinodor CQ in the RTgill-W1 cell line (first experiment).  

 

Effect concentrations expressed as mean measured concentration (mg/L)

 

Cytotoxicity 24 h (mg/L)

Predictedin vivoacute toxicity (LC50) (mg/L)

EC50

 4.76 (3.92 – 5.77) mg/L

1.98

LOEC

2.01 mg/L

n.a.

NOEC

< 2.01 mg/L

n.a.

 

Values in brackets are 95% confidence limits

n.a. not applicable

 

 

Effect concentrations, LOEC and NOEC of Sinodor CQ in the RTgill-W1 cell line (second experiment).

 

Effect concentrations expressed as mean measured concentration (mg/L)

 

Cytotoxicity 24 h (mg/L)

Predictedin vivoacute toxicity (LC50) (mg/L)

EC50

 4.29 (3.54 – 5.22) mg/L

1.79

LOEC

1.70 mg/L

n.a.

NOEC

0.97 mg/L

n.a.

 

Values in brackets are 95% confidence limits

n.a. not applicable

Validity criteria fulfilled:

yes

Conclusions:

The EC50 values based on mean measured concentrations in the RTgill-W1 cell assay were 4.76 mg/L and 4.29 mg/L in the first and second experiment, respectively (average 4.53 mg/L). Based on a regression equation developed with 37 fragrance molecules an in vivo LC50 of 1.98 mg/L and 1.79 mg/L, respectively, is predicted (mean LC50: 1.89 mg/L).

Sinodor CQ is a highly hydrophobic molecule with a log Kow value of 6.0. Its water solubility was determined to be only 0.421 mg/L (at 20°C in purified water). The LOEC, EC50 and LC50 values determined in this study are clearly above the water solubility of the chemical, and represent therefore a worst-case scenario.

The median of the under-/over-prediction (fold difference between LC50 predicted by the regression equation 1 in Appendix F of the attached report 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.

Effect concentrations expressed as mean measured concentration (mg/L)
Cytotoxicity 24 h (mg/L) Predicted in vivo acute toxicity (LC50) (mg/L)
EC50 4.76 (3.92 – 5.77) mg/L (exp 1) 1.98 (exp 1)
4.29 (3.54 – 5.22) mg/L (exp 2) 1.79 (exp 2)

EC50 (mean) 4.53 1.89

LOEC 2.01 mg/L (exp 1) n.a.
0.97 mg/L (exp 2) n.a.
< 2.01 mg/L (exp 1) n.a.

NOEC 0.68 mg/L (exp 2) n.a.

Executive summary:

SINODOR CQ is used exclusively 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.

The non-animal alternative method described herein, has been validated and published by the International Organization for Satndardization (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, is currently at an advanced stage of evaluation in the OECD Test Guideline Programme. 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.

The objective of the study was to determine the effects of Sinodor CQ 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. 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. The regression equation resulting from this study was used to predict the acute fish toxicity for Sinodor CQ.

 

The RTgill-W1 cells were seeded in 24-well plates and exposed in minimal medium to different Sinodor CQ 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).

 

Two experiments with the RTgill-W1 assay were performed using six different test concentrations. Concentrations ranged from 4 – 128 mg/L based on nominal concentrations in the first experiment (measured concentrations ranging from 3.71 – 33.32 mg/L at the start and 0.31 – 1.84 mg/L at the end of the exposure). In the second experiment, the test chemical was tested at 1 – 32 mg/L based on nominal concentrations (measured concentrations ranging from 1.07 – 15.26 mg/L at the start and 0.30 – 1.98 mg/L at the end of the exposure).

 

In the first experiment, the mean measured test chemical concentrations were 11.85, 17.58, 11.43, 7.90, 4.28, 2.01 mg/L (nominal concentrations 128, 64, 32, 16, 8, 4 mg/L). In the second experiment, mean measured concentrations were 8.41, 6.03, 3.04, 1.70, 0.97, 0.68 mg/L (nominal concentrations 32, 16, 8, 4, 2, 1 mg/L). The effect concentrations (EC50) are based on mean measured concentrations.

In order to predict a fish in vivo LC50 value from thein vitro EC50, a detailed benchmarking study on 38 fragrance chemicals was performed. 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 anin vivoLC50.

 

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

 4.76 (3.922 – 5.768) mg/L (exp 1)

4.29 (3.535 – 5.216) mg/L (exp 2)

1.98(exp 1)

1.79(exp 2)

LOEC

2.01 mg/L (exp 1)

1.70 mg/L (exp 2)

n.a.

NOEC

<2.01 mg/L (exp 1)

0.97 mg/L (exp 2)

n.a.

Values in brackets are 95% confidence limits

n.a. not applicable

 

In the first experiment, ≥10% cytotoxicity was observed at all tested concentrations, therefore the NOEC was considered to be <2.01 mg/L.After 24 hours, the EC₅₀ value based on mean measured concentration was 4.76 mg/L. Based on the regression equation derived from a training set of 37 fragrance molecules, an in vivo LC50 of 1.98 mg/L is predicted.

In the second experiment,the concentrations at which no significant (≤10%) dose related cytotoxicity occurred were 0.68 and 0.97 mg/L. The NOEC was thus considered to be 0.97 mg/L. After 24 hours, the EC₅₀ value based on mean measured concentration was 4.29 mg/L. Based on the regression equation an in vivo LC50 of 1.79 mg/L is predicted.

 

 

From the two experiments, the mean EC₅₀value and the mean predicted LC50 value were calculated. The final values are presented, below.

 

 

Mean effect concentrations (mg/L)

 

Cytotoxicity 24 h (EC50)

(mg/L)

Predictedin vivoacute toxicity (LC50) (mg/L)

Mean measured conc

 4.53

1.89

 

 

 

Conclusions

The mean EC50 value for Sinodor CQ was 4.53 mg/L, and the mean predicted LC50 value for fish was 1.89 mg/L based on measured concentrations. Since these values are above the water solubility, it is considered that this represents a worst case scenario.

Description of key information

SINODOR CQ is used exclusively in Cosmetics applications. In Europe, the testing of Cosmetics ingrdients 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.

The non-animal alternative method described herein, has been validated and published by the International Organization for Satndardization (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, is currently at an advanced stage of evaluation in the OECD Test Guideline Programme. 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.

The mean EC50 value for Sinodor CQ was 4.53 mg/L, and the mean predicted LC50 value for fish was 1.89 mg/L based on measured concentrations.

Since these values are above the water solubility, it is considered that this represents a worst case scenario.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.89 mg/L

Additional information

LC50 = 1.89 mg/L

NOEC = 0.97 mg/L