Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental phase conducted : 10 November 2014 through 14 November 2014
Reliability:
other: Currently non-guideline study. However, inter-laboratory ring study performed and in-reporting for submission to OECD for proposal of development of a new In vitro alternative test guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Principles of method if other than guideline:
Acute toxicity to fish is currently being tested according to OECD guideline 203. An alternative assay
recently adopted by the OECD is the fish embryo test (FET, guideline 236). The embryo test is still
costly and availability of embryos may be a limiting factor for early screenings. Since in fish, the gills
are the organs, which are most directly exposed to dissolved organic chemicals, acute toxicity often is
related to toxicity to the gills. A straightforward in vitro assay was thus developed by EAWAG (Swiss Center for Aquatic Research, Duebendorf, CH) based on cytotoxicity to a static monolayer culture of rainbow trout gill cell (RT-gill W1).

This assay allows reducing the use of vertebrate test animals significantly and it is currently undergoing detailed validation studies. An international ring-trial, in which our lab was a participating laboratory, showed this assay to be transferable and to produce highly reliable and reproducible results.

The objective of this study was to determine the 24-hour cytotoxicity of GR-50-3010 to the RTgill-W1
cell line. This in vitro model is particularly good to predict acute toxicity of chemicals with a narcotic
mode of action. A benchmarking study was performed on 38 fragrance chemicals with known fish
toxicity and the resulting regression equation was used to predict the acute fish toxicity for GR-50-3010.

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.
GLP compliance:
no

Test material

Specific details on test material used for the study:
Batch No. : GR-50-3010-3
Purity : 90.3% (sum of two main peaks)
Expiry : 04 November 2015

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
To confirm the concentration of GR-50-3010 in samples of test media during this test, at T0, 0.5 ml of
the samples aspirated from the test wells was diluted with 0.5 ml L15/ex. At T24, 1 ml samples from
the test wells were directly analysed. (At T0 no higher volume is available for analysis, while a higher
volume is used at T24 since substance concentrations are usually lower at this time point). These 1.0
ml samples were mixed in 2 ml glass vials with 400 μl of Methyl-tert-butylether (MTBE) containing as
internal standard 5 ppm of dodecane. Samples were extracted on a vortex mixer for 20 s, and then
ca. 200 μl of the supernatant was transferred to conical GC vials. 5 μl of these extracts were then
injected into a GC equipped with a flame-ionisation detector with a pulsed splitless injection.

Test solutions

Vehicle:
yes
Remarks:
Dimethyl Sulfoxide (DMSO)
Details on test solutions:
The test was conducted at 100, 50, 25, 12.5, 6.25 and 3.125 mg/L based on nominal concentrations
(66.38, 39.97, 18.03, 8.34, 4.43, and 2.21 mg/L based on mean measured concentrations). A control
group was also included.

Test organisms

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 (Duebendorf, Switzerland). Cells were seeded into cell culture flasks and
maintained in Leibovitz L15 medium amended with Glutamine not containing phenol red (Order nr.
21083; Gibco-Invitrogen; Basel, Switzerland). The medium was fortified with 5% foetal bovine serum
(FBS) and 1.1% of a penicillin Streptomycin solution. The cells were incubated at 19°C with normal air
(no CO2 enrichment) and 40% ventilation rate. The cells were split each week once with a 1:1 split.

Study design

Test type:
static
Water media type:
other: Leibovitz L15/ex medium
Limit test:
no
Total exposure duration:
24 h
Remarks on exposure duration:
A 24 hour exposure duration is the period described in the standard operating procedure of this cytotoxicity assay.

Test conditions

Test temperature:
19 °C
Reference substance (positive control):
yes
Remarks:
3,4-dichloroaniline

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
LC50
Remarks:
In vitro to In vivo extrapolation via regression
Effect conc.:
4.71 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Cell cytoxicity based on Alamar/PrestoBlue dye - cell viability with respect to mitochondrial respiration / metabolic activity
Duration:
24 h
Dose descriptor:
EC50
Remarks:
based on cell cytotoxicty before In vitro to In vivo extrapolation via regression
Effect conc.:
11.85 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Cell cytoxicity based on Alamar/PrestoBlue dye - cell viability with respect to mitochondrial respiration / metabolic activity.
Details on results:
After 24 hours, the concentration at which no significant (≤5%) dose related cytotoxicity occurred was 4.43 mg/L. The NOEC was thus considered to be 4.43 mg/L. After 24 hours the EC50 value based on mean measured concentration was determined to be 11.85 mg/L. Based on a regression equation derived from a training set of 38 fragrance molecules, an in vivo LC50 of 4.71 mg/L is predicted.

Chemical Analyses :
The results of the chemical analysis have been calculated by summing up the area of two peaks of the main components.

The limit of quantification (LOQ) for GR-50-3010 in medium using this method was 5 mg/L. Where samples contained levels below LOQ, measured values were extrapolated from analytical data obtained at the next higher test concentration.

The analytical sensitivity of the method applied to this test chemical with poor chromatographic behaviour at low concentrations is quite low. However, the key biological effect values (EC50 value) are determined from concentrations above the LOQ, thus this limited sensitivity of the analytical method for the lower concentrations does not affect the conclusions obtained.

Analysis of the test samples at 0 hours showed measured concentrations in the range from 2.7 mg/L to 85.4 mg/L. Analysis of the test samples after the 24 h incubation period showed measured concentrations in the range from 1.7 mg/L to 66.4 mg/L.
Results with reference substance (positive control):
Reference values for EC50 of 3,4-dichloroaniline :
a) Three independent studies in the test lab: 45.7 – 49.8 mg/L
b) Average values from 6 test labs in ring study: Average: 41.32; range 31.43 – 47.99 mg/L.

Conclusion
The EC50 was determined to be 43.3 mg/L based on nominal concentration after 24 h. The result of the international ring trial for EC50 is 31.43 mg/L – 47.99 mg/L after 24 h based on nominal concentration. Thus, these results were within the expected range.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Remarks:
The RT gil-W1 cell line assay is a non-guideline study for the time-being.
Executive summary:

After 24 hours, the concentration at which no significant (≤5%) dose related cytotoxicity occurred was

4.43 mg/L. The NOEC was thus considered to be 4.43 mg/L. After 24 hours the EC50 value based on

mean measured concentration was determined to be 11.85 mg/L. Based on a regression equation

derived from a training set of 38 fragrance molecules an in vivo LC50 of 4.71 mg/L is predicted.