fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Remarks:

as an alternative to the short-term toxicity for fish, a study according to OECD 249 was carried out with fish cells (RTgill-W1) in order to provide information to fill data gaps in the sense of a weight of evidence approach

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-08-05 to 2022-08-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Weight of evidence 4: Toxicity to fish gill cells via in vitro RTgill-W1 assay as alternative to the acute fish test.

Available acute tests with daphnia and algae with the test item, results from read-across analogue substances as well as QSAR predictions demonstrated that fish is not the most senstive species. Following the 3R princliples test with fish should be avoided when not necessary. Data indicated in this case that it is justified to use an alternative to the acute fish test. The RTgill-W1 assay according to OECD 249 was used.
Applicability: RTgill-W1 cell line assay-derived EC50 values have been demonstrated overall to be in excellent agreement with lethal concentrations (LC50 values) determined in fish acute toxicity tests for a wide range of chemicals (from OECD TG 249).
Data from other surfactants confirmed applicability; data for acute fish and corresponding data from RTgill-W1 cell line assay correlated very well (unpublished data).
Surfactants are not expected to be bioaccumulated via the food chain but via the aqueous phase. Surfactant uptake occurs primarily via the gills, therefore, a test with gill cells seems to be adequate.
Also the REACH Regulation supports the use of scientifically validated alterantive tests.
Article 25 (1) of the REACH Regulation states that testing on vertebrate animals shall only be performed as a last resort. Once scientifically validated according to internationally agreed validation principles (OECD GD 34 (OECD; 2005b)) in vitro test may fully or partly replace an in vivo test depending on the purpose for which the test method was validated and adopted.

Qualifier:

according to guideline

Guideline:

other: OECD Testguideline 249: Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay

Deviations:

no

GLP compliance:

yes

Remarks:

Chemical analysis was conducted under GLP. The cell assay was also conducted under GLP. However, it was carried out in process of establishing GLP within the facility. Therefore, there is no GLP certification at the point of signing this study report.

Analytical monitoring:

yes

Remarks:

Chemical analysis was conducted under GLP with quality assurance statement.

Details on sampling:

Concentrations were: 10.94, 21.88, 43.75, 87.50, 175.00, 350.00 mg/L, with an L-15/ex medium control included.
The highest concentration stock solution and the dilution series were prepared on the day of the exposure.
Cells were then exposed to 2.5 mL of the respective dosing solution in each well.
From each well, a sample of 500 μL was removed for chemical analysis and pooled per concentration in an autosampler vial.

Vehicle:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow trout (Oncorhynchus mykiss) gill cell line - RTgill-W1

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 evaluates toxicity photometrically by measuring the fluorescence of three indicator dyes. Resazurin (used as alamarBlue™), CFDA-AM and Neutral Red are used to measure metabolic activity, integrity of the cell membrane and integrity of the lysosome membrane, 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.
For each assay, an additional test is performed with a concentration range of 3,4-dichloroaniline, using a separate test plate for assay performance control.

RTgill-W1 cell line was used for this study (original stock obtained from Prof. N. C. Bols, University of Waterloo). 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 (for the test concentrations as well as 3,4-DCA assay performance positive control).
In all plates, wells A1 and A2 were not seeded to be left as the “cell-free controls”. Cells were left to attach and reach confluency for at least 24 h prior to starting the exposure. Cell culture passages P59 were used throughout the testing period.

Test type:

static

Water media type:

other: L-15/ex: is a protein free medium, containing the same amounts of salts, galactose and pyruvate as Leibovitz L-15 medium, used for exposure (“/ex”) of RTgill-W1 cells to a test chemical. L-15/ex is prepared according to Annex 3 to 5 of OECD 249.

Limit test:

no

Total exposure duration:

24 h

Test temperature:

19 ± 2 °C

Nominal and measured concentrations:

10.94, 21.88, 43.75, 87.50, 175.00, 350.00 mg/L
Measured concentrations were within ± 20% of nominal concentrations.

Details on test conditions:

RTgill-W1 cell line was used for this study (original stock obtained from Prof. N. C. Bols, University of Waterloo).
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 (for the test concentrations as well as 3,4-DCA assay performance positive control).
In all plates, wells A1 and A2 were not seeded to be left as the “cell-free controls”. Cells were left to attach and reach confluency for at least 24 h prior to starting the exposure. Cell culture passages P59 were used throughout the testing period.

Preparation of the stock solution and dilution series
As determined by a non-GLP range finding study, the exposure concentrations were 10.94, 21.88, 43.75, 87.50, 175.00, 350.00 mg/L, with an L-15/ex medium control included.
The highest concentration stock solution and the dilution series were prepared on the day of the exposure.
The highest concentration stock was prepared by weighing out 0.0398 g of the test item into a volumetric flask and made up to 100 mL of sterile deionized water. Each of the salt stocks required to prepare L-15/ex were then added in sequential order.
A serial dilution from the highest concentration stock (350 mg/L) was then carried out with a dilution factor of 2 (Table 2). The serial dilution was prepared under sterile conditions using autoclaved glassware. Extensive mixing between each dilution step was carried out by vortexing.

Table 2. Preparation of dilution series in L-15/ex
Name Concentration [mg/L] Volume L-15/ex [mL] Added vol. from solution Dilution factor

Stock 1 350.00 = stock solution

Stock 2 175.00 10 S1: 10 mL 2

Stock 3 87.50 10 S2: 10 mL 2

Stock 4 43.75 10 S3: 10 mL 2

Stock 5 21.88 10 S4: 10 mL 2

Stock 6 10.94 10 S5: 10 mL 2

Cell exposure and chemical sampling at 0 h
L-15 complete culture medium was removed from each well and all wells were washed with 1mL of L-15/ex prior to exposure. Cells were then exposed to 2.5 mL of the respective dosing solution in each well as detailed in the plate set-up.
From each well, a sample of 500 μL was removed for chemical analysis and pooled per concentration in an autosampler vial. The plates were then covered by adhesive foil and incubated for 24 h at 19 ± 2 °C (Incubator: Kt53 (3-40 °C), Binder, Tuttlingen, Germany) in the dark.
The samples for chemical analysis were stabilised by adding 0.6 mL from the pooled samples to 0.26 mL of the solvent mixture (acetonitrile (Scharlau) and 0.3 % formic acid (Sigma-Aldrich; provided by Arcardis Scweiz AG) in autosampler vials. This was prepared in duplicate for each test concentration and negative control. Samples were vortexed prior to storage at - 20 ± 4 °C.

Assessment of cell viability
Cell viability was measured at the end of the 24 h exposure according to the OECD TG249. Prior to the cell viability assessment, samples of the exposure medium were taken from the test item exposure plate for chemical analysis as described in Section 3.3.
Cells were first visually inspected during the phosphate buffered saline (PBS; 1 mL per well) wash of the cells. The wash solution was replaced with the 400 μL of the Resazurin/CFDA-AM working solution and incubated on the cells for 30 min at 19 ± 2 °C. The fluorescence of the Resazurin-based dye (exc: 530 nm, em: 595 nm), and immediately following the fluorescence of CFDA-AM (exc: 493 nm, em: 541 nm), were measured as raw fluorescent units with a plate reader (Synergy H1, Agilent, United States).
The Resazurin/CFDA-AM working solution was then replaced with 400 μL of the Neutral Red working solution in all wells. After 60 min of incubation at 19 ± 2 °C in the dark, the Neutral Red working solution was discarded and the cells were washed with 400 μL fixative solution. The fixative was then replaced in all wells by 400 μL of extraction solution. After a 10 min incubation on a plate shaker at room temperature, the fluorescence of Neutral Red (exc: 530 nm, em: 645 nm) was measured as raw fluorescent units with the fluorescent plate reader.

Data analysis
All data analysis was carried out using the Gen5© secure software (Agilent, United States). Firstly, the raw fluorescence data (arbitrary units) gained was used to calculate the toxicity as a percentage of cell viability compared to the respective control (L-15/ex). As the positive control was dissolved in DMSO, the organic solvent control was used as the reference. For the test item plate, the L-15/ex negative control was used as the reference. The corresponding average “cell-free control” fluorescence units were subtracted from the fluorescence units of each well of the corresponding test plate.
For each replicate well of each chemical test concentration, the percentage of cell viability with regard to the respective control was calculated. Therefore, the average of the fluorescence units of the control was set to 100 % (indicating that 100 % of the cells are viable) and corresponding cell viability calculated as shown by formula 1:
% 𝑐𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦=(𝑓𝑙𝑢𝑜.𝑢𝑛𝑖𝑡𝑠 𝑐ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝑥 100%) / 𝑓𝑙𝑢𝑜.𝑢𝑛𝑖𝑡𝑠 𝑟𝑒𝑠𝑝𝑒𝑐𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 (1)

Calculation of the average and standard deviation of the % cell viability per chemical test concentration was then carried out, with the % cell viability used to determine the concentration-response curve. Non-linear regression sigmoidal concentration−response curve fitting was carried out, with constrains set for Bottom (0.0) and Top (100.0), from which the EC50 values were calculated.

Reference substance (positive control):

yes

Remarks:

3,4-Dichloroaniline As per the OECD TG249, a positive control must be carried out for each run of the study. This involved exposing a plate seeded with RTgill-W1 to 3,4-Dichloroaniline (3,4-DCA) in parallel to the plates exposed to the test item.

Key result

Duration:

24 h

Dose descriptor:

EC50

Remarks:

alamarBlue™

Effect conc.:

211.38 mg/L

95% CI:

149.46 - 273.3

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

100% UVCB

Basis for effect:

other: metabolic activity

Remarks on result:

other: Resazurin used as alamarBlue™ to measure metabolic activity

Key result

Duration:

24 h

Dose descriptor:

EC50

Remarks:

Neutral Red

Effect conc.:

303.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

100% UVCB

Basis for effect:

other: lysosome integrity

Remarks on result:

other: Neutral Red diffuses into the cells and accumulates in lysosomes. Disruption of lysosomes therefore results in a decrease in Neutral Red fluorescence.

Key result

Duration:

24 h

Dose descriptor:

EC50

Remarks:

CFDA-AM

Effect conc.:

311.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

100% UVCB

Basis for effect:

other: membrane integrity

Remarks on result:

other: CFDA-AM is an esterase substrate that is converted to 5- carboxyfluorescein in intact cells where it is retained. A decline in CFDA-AM fluorescence therefore indicates disturbance of plasma membrane integrity.

Duration:

24 h

Dose descriptor:

EC50

Remarks:

alamarBlue™

Effect conc.:

139.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: main component

Basis for effect:

other: metabolic activity

Remarks on result:

other: Resazurin used as alamarBlue™ to measure metabolic activity

Duration:

24 h

Dose descriptor:

EC50

Remarks:

Neutral Red

Effect conc.:

199.77 mg/L

Nominal / measured:

nominal

Conc. based on:

other: main component

Basis for effect:

other: lysosome integrity

Remarks on result:

other: Neutral Red diffuses into the cells and accumulates in lysosomes. Disruption of lysosomes therefore results in a decrease in Neutral Red fluorescence.

Duration:

24 h

Dose descriptor:

EC50

Remarks:

CFDA-AM

Effect conc.:

205.45 mg/L

Nominal / measured:

nominal

Conc. based on:

other: main component

Basis for effect:

other: membrane integrity

Remarks on result:

other: CFDA-AM is an esterase substrate that is converted to 5- carboxyfluorescein in intact cells where it is retained. A decline in CFDA-AM fluorescence therefore indicates disturbance of plasma membrane integrity.

Details on results:

Measured concentrations were within ± 20% of nominal concentrations. Therefore, the EC50 concentrations are reported as nominal concentration of test item and main component (65.9 %).

The assay allows the detection of three cell viability measures on the same set of cells, indicating cellular (i.e. cyto-)toxicity. It uses a combination of the following fluorescent indicator dyes: a
Resazurin-based dye for measuring cell metabolic activity (alamarBlue™); 5-carboxyfluorescein diacetate acetoxy methyl ester (CFDA-AM) for assessing the integrity of the cell membrane; and Neutral Red, which evaluates the integrity of the lysosomal membrane.
The assay is evaluated by measuring the fluorescence of these dyes in the medium and the results are expressed as percent cell viability in comparison to an untreated control group.

Results with reference substance (positive control):

Dose response curves covering the 50% effect level were obtained for all three fluorescent indicator dyes, with data plotted based on nominal chemical concentrations (product based). The EC50 with confidence levels were calculated for each of the fluorescent indicators.

AlamarBlue™ CFDA-AM Neutral Red
EC50 (mg/L) 48.34 60.13 52.18

Validity criteria fulfilled:

yes

Conclusions:

According to the outcome of the OECD TG 249 with rainbow trout gill cell line RTgill-W1, the EC50 value of the most senstitive endpoint (alamarBlue™ => metabolic activity) is 211.38 mg/L. This indicates a rather low toxicity of the test item towards fish.

Executive summary:

Study:
Use of the RTgill-W1 cell line assay to predict the acute toxicity (with chemical analysis)
Guideline following: OECD TG249

Test concentrations:
Control, 10.94, 21.88, 43.75, 87.50, 175.00, 350.00 mg/L
Test System:
Rainbow trout (Oncorhynchus mykiss) gill cell line - RTgill-W1
Exposure conditions:
RTgill-W1 cells were exposed in L-15/ex exposure medium at 19 ± 1°C
Exposure duration:
24 hours

Overview final test result:

	AlamarBlue™	CFDA-AM	Neutral Red
EC50 (mg/L) (nominal concentration of test item)	211.38	311.76	303.14
EC50 (mg/L) (nominal concentration of main component (65.9 %))	139.30	205.45	199.77

The test item is a UVCB substance, therefore the effect concentrations are derived from the nominal concentration of the test item, as the measured concentrations were within ± 20% of the nominal concentrations. 

Article 25 (1) of the REACH Regulation states that testing on vertebrate animals shall only be performed as a last resort. Once scientifically validated according to internationally agreed validation principles (OECD GD 34 (OECD; 2005b)) in vitro test may fully or partly replace an in vivo test depending on the purpose for which the test method was validated and adopted.

 

 

Reference 2

Endpoint:

short-term toxicity to fish

Remarks:

Weight of evidence 1: Intrinsic substance properties and QSAR to predict short-term toxicity to fish

Type of information:

(Q)SAR

Remarks:

QSAR Toolbox (v4.5) and EPI Suite (v4.11)

Adequacy of study:

weight of evidence

Study period:

2022

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Weight of evidence 1: Intrinsic substance properties and QSAR to justify the use of the RTgill-W1 assay as alternative to the acute fish test.

Qualifier:

no guideline required

Principles of method if other than guideline:

Use of EPI suite (v4.11) and therein ECOSAR Version 1.11

Remarks on result:

other: The ECOSAR predictions indicated that fish is the least sensitive species compared to daphnia and algae.

Conclusions:

The intrinsic substance properties indicate rather a baseline toxicity of the test item. The ecotoxic effects of surfactants are generally considered to be nonspecific. The ECOSAR predictions indicated that fish is the least sensitive species compared to daphnia and algae. Daphnia seems to be the most susceptible towards the test item. This justifies the use of the alternative approach the RTgill-W1 assay instead of acute fish test and goes in line with REACH regulation to use animal testing as last resort.

Article 25 (1) of the REACH Regulation states that testing on vertebrate animals shall only be performed as a last resort. Once scientifically validated according to internationally agreed validation principles (OECD GD 34 (OECD; 2005b)) in vitro test may fully or partly replace an in vivo test depending on the purpose for which the test method was validated and adopted.

Executive summary:

The following endpoint specific predictions have been obtained by the QSAR Toolbox

Endpoint Specific
DNA binding by OASIS		No alert found
Protein binding by OECD		No alert found
Acute aquatic toxicity MOA by OASIS		Basesurface narcotics
Acute aquatic toxicity classification by Verhaar (Modified)		Class 5 (Not possible to classify according to these rules)
Bioaccumulation - metabolism half-lives		Very fast
Bioaccumulation - metabolism alerts		-CH-   [linear] -CH2-  [linear] Amide   [-C(=O)-N  or -C(=S)-N] Linear C4 terminal chain  [CCC-CH3] Methyl  [-CH3]
DNA alerts for AMES, CA and MNT by OASIS		No alert found
Aquatic toxicity classification by ECOSAR		Acid moiety Amides Salt
Biodegradation fragments (BioWIN MITI)		-CH-   [linear] -CH2-  [linear] Aliphatic acid   [-C(=O)-OH] Amide   [-C(=O)-N  or -C(=S)-N] Methyl  [-CH3]

The intrinsic substance properties indicate rather a baseline toxicity of the test item. The ecotoxic effects of surfactants are generally considered to be nonspecific, with exposure leading to disruption of biological membrane integrity (Boeije et al. 2006). 

Surfactants are not expected to be bioaccumulated via the food chain but via the aqueous phase. Surfactant uptake occurs primarily via the gills. Thus, evidence is given to use the RTgill-W1 assay as alternative to the acute fish test. Especially, with regard to the 3R principle and to safe animal life. Bioconcentration of organic chemicals is driven by hydrophobicity as tendency to escape from water (Tolls, 1998). It appears that bioconcentration of surfactants increases with increasing length of the acyl/alkyl chain, but their bioconcentration is not related to lipid content and thus surfactants are not stored in the fish.

In both ECOSAR predictions, fish are indicated as the least sensitive organisms relative to other trophic levels to the test item. Daphnia seems to be the most sensitive species.

Results from ECOSAR for main component (neutral non-salt form of the molecule for proper estimation)

ECOSAR Class Organism Duration End Pt mg/L (ppm)
=========================== ================== ======== ====== ==========
--> Acid moeity found: Predicted values multiplied by 10

Amides -acid                : Fish 96-hr LC50                 88.212
Amides -acid                : Daphnid 48-hr LC50           49.454
Amides -acid                : Green Algae 96-hr EC50       3.805
Amides -acid                : Fish (SW) 96-hr LC50          79.081
Amides -acid                : Mysid (SW) 96-hr LC50         7.939
=========================== ================== ======== ====== ==========
Neutral Organic SAR     : Fish 96-hr LC50                  13.084
(Baseline Toxicity)        : Daphnid 48-hr LC50              8.505
                                  : Green Algae 96-hr EC50       11.083

 

 

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

read-across from similar mixture/product

Remarks:

available test results from algae and daphnia

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Justification for type of information:

Weight of evidence 3: Available experimental studies on other trophic levels, algae and daphnia to justify the use of the RTgill-W1 assay as alternative to the acute fish test.

Qualifier:

no guideline required

Principles of method if other than guideline:

Comparison of the outcome of the algae and daphnia test with the test item to make assumptions on toxicity towards fish.

GLP compliance:

yes

Duration:

72 h

Dose descriptor:

EC50

Remarks:

growth inhibition algae

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition algae

Remarks on result:

other: This is the results of the short-term algae test.

Duration:

48 h

Dose descriptor:

EC50

Remarks:

immobility daphnia

Effect conc.:

32.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobility of invertebrates

Remarks on result:

other: This is the result of the short-term invertebrate test

Conclusions:

Based on effects on organisms of other trophic levels it seems that invertebrates are the most sensitive test organisms towards the test item. This gives evidence and justify the use of the RTgill-W1 assay as alternative test system to the acute fish test in order to save animal life, follow the 3R principles and the REACH regulation to use animals as last resort.

Article 25 (1) of the REACH Regulation states that testing on vertebrate animals shall only be performed as a last resort. Once scientifically validated according to internationally agreed validation principles (OECD GD 34 (OECD; 2005b)) in vitro test may fully or partly replace an in vivo test depending on the purpose for which the test method was validated and adopted.

Executive summary:

The results from algae and daphnia test with the test item are as follows:

Short-term toxicity to invertebrates	EC50 = 32.9 mg/L (test mat. geometric mean))
Toxicity to aquatic algae	EC50 >100 mg/L (test mat.) EC10 = 51.2 mg/L (test mat.)

The ratio of toxicity to daphnia vs. algae is below 0.5 (=> 33/100 = 0.33). Daphnia has a considerable lower tolerance to the substance than algae. In this case, the substance mode of action is assumed to be rather specific to invertebrates. This can be an indication that the substance will most likely have a low toxicity to fish (Moe et al. 2020).

The trophic levels represented by algae and daphnia have been shown to be more sensitive than the acute fish test (Hutchinson et al. 2003; Jeram et al. 2005) and fish embryo test (Rawlings et al. 2019) between 75% to 80% of the time. Given that hazard classification and environmental risk assessment are based on the most sensitive taxa, algae and daphnids tend to routinely drive these assessments (Lillicrap et al. 2020).

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990-01-22 to 1990-03-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Weight of evidence 2: Read-across Analogue substances to justify the use of the RTgill-W1 assay as alternative to the acute fish test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substances was directly added to the test vessels.

Test organisms (species):

Leuciscus idus

Details on test organisms:

TEST ORGANISM
- Common name: orfe
- Source: Fish farm Eggers, Hohenwestedt
- Age at study initiation: approx. 4 weeks
- Corpulence factor: 0.8 - 1.1 g/cm^3
- Feeding during test: none

ACCLIMATION
- Acclimation period: For at least 7 days
- Acclimation conditions: same dilution water
- Type and amount of food: Pitti Floxi-Flocken Alleinfutter für Zierfische
- Feeding frequency: twice a week

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

15 ± 3° dH (total hardness)

Test temperature:

20 ± 0.5 °C

pH:

7.3 - 8.2

Dissolved oxygen:

4.4 - 8.6 mg/L

Nominal and measured concentrations:

Nominal concentration: 150, 175, 190 and 200 mg/L (based on test material)
Nominal concentration: 48, 56, 60.8 and 64 mg/L (based on active matter)

Details on test conditions:

TEST SYSTEM
- Test vessel: tanks (L 28.5 cm / W 21.0 cm / H 24.0 cm)
- Material: glass; Fill volume: 15 L
- Aeration: weak aeration during test period
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 3.7 fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mixture of drinking water and deionized water
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen concentration and pH were measured after 24, 48, 72 and 96 h test duration.

EFFECT PARAMETERS MEASURED: Mortality was recorded after 24, 48, 72 and 96 h test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 1.1

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

62.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: converted to active matter

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

195 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

190 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: none

Validity criteria fulfilled:

yes

Conclusions:

The LC50 of the test item was found to be 62.4 mg/L a.i.

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008-08-21 to 2008-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Weight of evidence 2: Read-across Analogue substances to justify the use of the RTgill-W1 assay as alternative to the acute fish test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: colourless liquid
Purity: 29.8% dry content

Analytical monitoring:

yes

Details on sampling:

0, 96h

Vehicle:

no

Details on test solutions:

direct weighing, limit test at 100 mg/L test item (29.8 mg/L a.i.)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: Aquarium am Aegi, Hannover. All fish used in the test originated from the same delivery of the supplier
- Length at study initiation (length definition, mean, range and SD): 2.80 cm
- Weight at study initiation (mean and range, SD): 0.21 g

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): 23 ± 2 °C and diffuse light. Water change, once per week. dissolved oxygen more than 80% of air saturation value.
- Type and amount of food during acclimation: 4% of fish body weight per feeding day, Stör perlets; SERA GmbH
- Feeding frequency during acclimation: 3 times per week
- Health during acclimation (any mortality observed): < 5% mortality, no desease treatments were administered
Fish were not fed during test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness 59 mg/L as CaCO3

Test temperature:

23 ± 2 °C

pH:

7.23-7.62

Dissolved oxygen:

Oxygen saturation 95-100%

Nominal and measured concentrations:

nominal 100 mg/L test item concentration, 16.6 mg/L DOC of test media

Details on test conditions:

7 zebra fish were used per limit concentration and control.
10 L per vessel, gentle aeration was provided.
Glass aquaria loosley covered by glass tops
Fish density was less than 1 g per fish per liter test solution.
Fish were randomly introduced to individual replicates.

pH-value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h.
Observations were made after 24, 48, 72, and 96 h.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 29.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

29.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

29.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Validity criteria on Oxygen saturation (>= 60%) and Mortality in the control (0%) were met.

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item did not cause any effects to zebra fish after 96 h when tested with a nominal concentration of 100 mg/L (29.8 mg/L a.i.). The NOEC (0-96 h) is laid down as 100 mg/L (29.8 mg/L a.i.).

Executive summary:

In this static limit test according OECD 203 the test item Hostapon SG (a.i content of solution 29.8% ww) did not cause any effects to zebrafish after 96h when tested with a nominal concentration of 100 mg/L (29.8 mg/L a.i.). LC0 and NOEC are 100 mg/L (29.8 mg/L a.i.). LC100 is > 100 mg/L (> 29.8 mg/L a.i.).

Validity criteria on Oxygen saturation (>= 60%) and Mortality in the control (0%) were met.

Reference 6

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Read-across is used in this weight of evidence approach to support the justification for the use of an alternative approach for the acute fish test.

Adequacy of study:

weight of evidence

Justification for type of information:

Weight of evidence 2: Read-across Analogue substances to justify the use of the RTgill-W1 assay as alternative to the acute fish test.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

LC50

Remarks:

Sodium N-cocoyl glutamate

Effect conc.:

195 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Remarks:

Sodium N-cocoyl glutamate

Effect conc.:

62.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Remarks:

Sodium N-cocoyl glycinate

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Remarks:

Sodium N-cocoyl glycinate

Effect conc.:

> 29.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Conclusions:

When comparing the short-term test results of all three analogue substances of the three trophic levels of aquatic organisms there is a clear indication that fish is always the least sensitive organism to the test items. Daphnia seems to be the most senstive organism to these group of compounds.

Executive summary:

When comparing the short-term test results of all three analogue substances of the three trophic levels of aquatic organisms there is a clear indication that fish is always the least sensitive organism to the test items. Daphnia seems to be the most sensitive organism to these group of compounds.

	Target chemical	Source chemicals
Chemical name	Disodium lauroyl-glutamate	Sodium cocoyl glycinate	Disodium N-cocoyl glutamate
Short-term toxicity to fish	EC50 = 211.38 mg/L (test mat.) OECD 249 RTgill-W1 assay	LC50 > 100 mg/L (test mat.) LC50 > 29.8 mg/L (a.i.)	LC50 = 195 mg/L (test mat.) LC50 = 62.4 mg/L (a.i.)
Short-term toxicity to invertebrates	EC50 = 32.9 mg/L (test mat. geometric mean))	EC50 = 6.5 mg/L (a.i.)	EC50 = 18 mg/L (test mat.)
Toxicity to aquatic algae	EC50 >100 mg/L (test mat.) EC10 = 51.2 mg/L (test mat.)	EC50 = 61 mg/L (a.i.) EC10 = 21.7 mg/L (a.i.)	(-)

This justifies to use the RTgill-W1 assay as alternative to the acute fish test in line with animal safety, 3R principle and REACH regulation to use animal tests only as last resort.

Article 25 (1) of the REACH Regulation states that testing on vertebrate animals shall only be performed as a last resort. Once scientifically validated according to internationally agreed validation principles (OECD GD 34 (OECD; 2005b)) in vitro test may fully or partly replace an in vivo test depending on the purpose for which the test method was validated and adopted.

Description of key information

An alternative study according to OECD 249 with rainbow trout gill cells with the test item has been performed.
EC50 (24h) = 211.38 mg/L (nominal) for Oncorhynchus mykiss gill cells (OECD 249).

	AlamarBlue™	CFDA-AM	Neutral Red
EC50 (mg/L) (nominal concentration of test item)	211.38	311.76	303.14
EC50 (mg/L) (nominal concentration of main component (65.9 %))	139.30	205.45	199.77

The weight of evidence approach with argumentation lines 1-4 provide evidence and justifies the use of an alternative test system (RTgill-W1 assay) for the acute fish test following the 3R principles since the fish is not the most sensitive organism relative to other trophic levels to the test item.

WoE Line 1: The intrinsic substance properties indicate rather a baseline toxicity of the test item. The ecotoxic effects of surfactants are generally considered to be nonspecific. The ECOSAR predictions indicated that fish is the least sensitive species compared to daphnia and algae. Daphnia seems to be the most susceptible towards the test item. This justifies the use of the alternative approach the RTgill-W1 assay instead of acute fish test and goes in line with REACH regulation to use animal testing as last resort.

WoE Line 2: Two studies from structural analogue substances are available and confirm the approach. In studies with both test items invertebrates (daphnia) are the most sensitive organisms and not fish.

WoE Line 3: Based on effects on organisms of other trophic levels it seems that invertebrates are the most sensitive test organisms towards the test item. This goes in line with QSAR predictions and results of structural analogues of the test item. This gives evidence and justify the use of the RTgill-W1 assay as alternative test system to the acute fish test in order to save animal life, follow the 3R principles and the REACH regulation to use animals as last resort.

WoE Line 4: According to the outcome of the in vitro test OECD TG 249 with rainbow trout gill cell line RTgill-W1, the EC50 value of the most sensitive endpoint (alamarBlue™ => metabolic activity) is 211.38 mg/L. This indicates a rather low toxicity of the test item towards fish.

Taken together, predictions, comparisons with structurally similar substances and experiments have clearly shown that fish are the least sensitive to the test item compared to other trophic levels. This allows, on the basis of scientifically based results in the sense of animal welfare and 3R principle, to avoid killing fish for an acute fish test and to use instead a recognized OECD guideline 249 for an in vitro test with RTgill-W1 cells.

Also the REACH Regulation supports the use of scientifically validated alterantive tests.
Article 25 (1) of the REACH Regulation states that testing on vertebrate animals shall only be performed as a last resort. Once scientifically validated according to internationally agreed validation principles (OECD GD 34 (OECD; 2005b)) in vitro test may fully or partly replace an in vivo test depending on the purpose for which the test method was validated and adopted.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC50

Remarks:

alamarBlue™

Effect concentration:

211.38 mg/L