Sodium hydrogen N-(1-oxotetradecyl)-L-glutamate

Additional ecotoxological information

Administrative data

Endpoint:

additional ecotoxicological information

Remarks:

RTgill-W1 cell line assay to predict acute fish toxicity according to ISO Standard 21115 Edition 1

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Used as bridging study to support Read Across for acute fish toxicity

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ISO standard 21115

Version / remarks:

2019

Deviations:

yes

Remarks:

without chemical analysis

GLP compliance:

not specified

Type of study / information:

The RTgill-W1 (rainbow trout (Oncorhynchus mykiss) gill) cell line assay allows the detection of
three toxicity endpoints with the same set of cells. The assay is evaluated photo-metrically by
measuring the fluorescence of three indicator dyes. AlamarBlue, CFDA-AM and Neutral Red (NR)
are used to measure metabolic activity, integrity of the cell membrane and integrity of the lysosome membranes, respectively. The results are expressed as % cell viability compared to an
untreated control.
The compounds are tested in a dilution series and the EC50 value of each test compound is
calculated from the results. Extensive studies2-6 have shown that the cell line-derived results
agree very well with data from the acute fish test (LC50) with the exception of very specifically
acting (neurotoxic) compounds.
For each round of testing, an additional test is performed with a concentration range of 3,4-Dichloroaniline (3,4-DCA) in defined, standard cell exposure medium (L-15/ex) using a separate
test plate for assay performance control

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen N-(1-oxotetradecyl)-L-glutamate

EC Number:

253-981-4

EC Name:

Sodium hydrogen N-(1-oxotetradecyl)-L-glutamate

Cas Number:

38517-37-2

Molecular formula:

C19H35NO5.Na

IUPAC Name:

sodium hydrogen N-(1-oxotetradecyl)-L-glutamate

Details on test material:

- Name of test material (as cited in study report): Sodium hydrogen N-(1-oxotetradecyl)-L-glutamate
- Molecular formula (if other than submission substance): C19H35N1O5
- Molecular weight (if other than submission substance): 357.49
- Smiles notation (if other than submission substance): O=C(NC(C(=O)O)CCC(=O)O)CCCCCCCCCCCCC
- Analytical purity: not applicable

Specific details on test material used for the study:

Batch no.: 98 supplied by the sponsor
Aspect: clear liquid, colourless to yellowish
Purity: 29.8%
Water solution
Storage condition of test material: room temperature (15 °C - 25 °C), stable for at least 12 months
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium): stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
More information can be found on the attached report

Results and discussion

Any other information on results incl. tables

Before starting the final test, a range-finding test was performed to obtain the optimal test item concentrations for the final RTgill-W1 test. Dilution series of six test item concentrations (product based), from 3.2 - 10000 mg/L (dilution factor 5), were tested. Effects on cell viability were observed at 130.8 mg/L (product based) and higher for all three indicator dyes. Based on these
results, the test item concentration range for the final test performance was defined as displayed in Table 3 of the attached report. Appendix A2 of the attached report summarizes the results of the range-finding test.
RTgill-W1 cells at a density of 350'000 cells per ml were seeded into wells of one 24-well plate(Greiner Bio-One, Frickenhausen, Germany) per item to test and for the 3,4-DCA assay performance (positive control). Cells were left to attach and reach confluency within 48 hours prior to starting the exposure. Cell culture passages P 62-64 were used for the test.
Preparation of the stock solution and dilution series
The test item stock solution and the dilution series were prepared on the day of starting the exposure. Tables 2 and 3 of the attached report illustrate the steps performed to prepare for the test. Appendix 2 of the attched report summarizes the procedure for the assay performance control, 3,4-DCA (concentration range 100.00mg/L - 3.125 mg/L, dilution factor 2).  RTgill-W1 cells in 24 well plates (see 5.1) were exposed according to ISO 21115 (see Figure 1 of the attched report).
The exposure duration was 24 hours at 19 ± 1°C (Incubator: BK-700, (3-40 °C) Heraeus Instruments, Hanau, Germany). Cell viability was measured according to ISO 21115 using the cell viability indicator dyes (see 3) and a fluorescent plate-reader (Infinity M200, Tecan, Männedorf, Switzerland).
Exposure medium samples were taken at 0 h (0.5 ml) and 24 h (1 ml) of exposure directly from each of the wells. All medium samples were stored at -20 ºC.
Fluorescent readings from cell viability assays are recorded as fluorescent units. The average background fluorescence of a cell-free control is subtracted from the fluorescent values of the cell-containing wells. The resulting values are averaged across triplicate wells for each of the conditions (control, different chemical exposure concentrations). These average values are then
expressed as “% cell viability” compared to the control, which is set to 100 %. The resulting concentration-response curves were used to determine the chemical concentration that caused a 50 % reduction in cell viability (EC50) based on nonlinear regression sigmoidal concentration−response curve fitting, using the Hill slope equation of GraphPad Prism 8.
One-way ANOVA, followed by Dunnet’s test, was applied to determine the No-Observed-Effect Concentration (NOEC).
The non-toxic concentration (NtC) is derived based on a combination of response-curve modelling and statistical means. It was calculated using the open-source algorithm published in (https://utox.shinyapps.io/NtC_NtC/). All validity criteria as described on the report attached were respected. The table below shows the results in % of active ingredient corrected according to purity.

Values active ingredient	AlamarBlue	CFDA-AM	Neutral Red
EC50 mg/l	57.07	73.93	56.23

 

Applicant's summary and conclusion

Conclusions:

The lowest cell-line derived EC50 value (product based) was obtained for Neutral Red being 56.23 mg/L (on active ingredient)

Executive summary:

In vitro studies using the RTgill-W1 cell line assay have been used to predict the toxicity to fish for the source and targe substances according to ISO Standard 21115 (OECD Test Guideline 249, published June 2021).  The RTgill-W1 cell line is derived from rainbow trout and allows the detection of three toxicity endpoints with the same set of cells.  The assay is evaluated photometrically by measuring the fluorescence of three indicator dyes.  AlamarBlue, CFDA-AM and Neutral Red (NR) are used to measure metabolic activity, integrity of the cell membrane and integrity of the lysosome membranes, respectively.  In each case a reduction in the fluorescence indicates a disruption to the metabolic activity, cell membrane integrity or lysosome membrane integrity.  The results are expressed as % cell viability compared to an untreated control.  The compounds are tested in a dilution series and the EC₅₀ value of each test compound is calculated from the results.  Extensive studies have shown that the cell line-derived results agree very well with data from the acute fish test (LC₅₀) with the exception of very specifically acting (neurotoxic) compounds.  For each round of testing, an additional test was performed with a concentration range of 3,4-Di-chloroaniline (3,4-DCA) in defined, standard cell exposure medium (L-15/ex) using a separate test plate for assay performance control.  One-way ANOVA, followed by Dunnett’s test, was applied to determine the NOEC.  The non-toxic concentration was derived based on a combination of response-curve modelling and statistical means.

For the target substance, the cells were exposed for 24 h at 19 ± 1 °C to test concentrations of 125 – 4 000 mg/L.  When corrected for purity the EC₅₀ was determined to be 57.07 mg/L with AlamarBlue, 73.93 mg/L with CFDA-AM and 56.23 mg/L with neutral red.

For the source substance, DSCG, the cells were exposed for 24 h at 19 ± 1 °C to test concentrations of 18.75 - 600 mg/L.  When corrected for purity the EC₅₀ was determined to be 60.21 mg/L with AlamarBlue, 87.51 mg/L with CFDA-AM and 49.74 mg/L with neutral red.  Therefore, the lowest cell-line derived EC₅₀ value was obtained for Neutral Red and toxicity for source and target can be evaluated as equivalent.