Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 March 2020 - 04 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2nd edition)

Version / remarks:

Adopted February 08, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil
Chemical name: 2,2-bis[({3-[methyl(phenyl)amino]propanoyl}oxy)methyl]butyl 3-[methyl(phenyl)amino]propanoate
EC number: 951-458-1

Analytical monitoring:

yes

Details on sampling:

During the final test, samples for possible analysis were taken from all test concentrations and the controls according to the schedule below. Stock solutions in ACN used for preparation of test solutions were sampled as well. The method of analysis is described in the appended Analytical Report (Appendix 4).

Frequency at t=0 h, t=24 h and t=72 h.
Volume 1.0 mL from the approximate centre of the test vessels.
Storage Samples taken from the test solutions were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling. Samples taken from the stock solutions were kept at room temperature until the next set of aquatic samples was transferred to the analytical laboratory at the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a loading rate of 3.2 µg /L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Remarks:

Acetonitrile

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Preparation of Test Solutions
The batch of C894 tested was a light orange liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Due to the indicated light-sensitivity of the test item, preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil where possible to minimize exposure to light.

Range-finding test
Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. To this end, the according test item amounts were weighed onto watch discs and added to a 1 L flasks. Subsequently, 1 L test medium was added to each flask and a three-day period of magnetic stirring applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Full tests
Based on the results obtained in the range-finding and the saturation test, Water Accommodated Fractions (WAFs) were prepared by using stock solutions in acetonitrile (ACN; Fisher Chemical, Geel, Belgium) with nominal concentrations of 0.050, 0.090, 0.16, 0.28 and 0.50 µg/mL. Empty test vessels were individually spiked with 1.0 mL of the respective stock. Each replicate of the solvent control received 1.0 mL ACN. The solvent was completely evaporated overnight before addition of the test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent. Thereafter, 50 mL test medium was added to each vessel. Test solutions, including those of the solvent control, were agitated for a period of one day to ensure maximum dissolution of test item in test medium. Vessels were sealed during agitation to prevent evaporation of test medium. All test solutions were clear and colourless at the end of the preparation procedure. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Any residual volumes were discarded.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
Common name: Raphidocelis subcapitata
Strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Age of inoculum: Not reported

ACCLIMATION
Acclimation period: Not reported

FRESHWATER ALGAE CULTURE
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

According to OECD guidleine

Post exposure observation period:

No post exposure observation

Hardness:

(Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

20 °C

pH:

8.1 ± 0.2

Dissolved oxygen:

Not reported

Salinity:

Not reported - freshwater

Conductivity:

Not reported - freshwater

Nominal and measured concentrations:

WAFs individually prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 µg/L.

Details on test conditions:

TEST SYSTEM
Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Initial cells density: 1 x 10^4 cells/mL
No. of vessels per concentration: 3 replicates of each test concentration
No. of vessels per control: 6 replicates of each control
No. of vessels other test vessels: 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae


GROWTH MEDIUM
Standard medium used: yes


STOCK CULTURE MEDIUM
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test.
The cell density was measured immediately before use.
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:

NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2


OTHER TEST CONDITIONS
Sterile test conditions: Not reported
Adjustment of pH: No
Photoperiod: Not reported
Light intensity and quality: Continuously using TLD-lamps with a light intensity of 85 µE.m-2.s-1.


EFFECT PARAMETERS MEASURED
Determination of cell concentrations: microscope and counting chamber


TEST CONCENTRATIONS
RANGE-FINDING TEST
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three replicates of exponentially growing algal cultures were exposed to WSFs individually prepared at loading rates of 1.0, 10 and 100 mg/L and to a control.
- Cell densities were recorded only at the end of the exposure period.
- One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- pH was only measured in the control and the highest test concentration.
- At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

FINAL TEST
WAFs individually prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 µg/L.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENTS AND RECORDINGS
pH At the beginning and at the end of the test, for all test concentrations and the control
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the final test, microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

RECORDING CELL DENSITIES
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

CONTROLS
Test medium without test item or other additives (blank control) and test medium without test item but with the additive used in the treatment of the stock solutions (solvent control).

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 6.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

6.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Reported statistics and error estimates:

Statistical comparison of algal growth between the blank and solvent control showed that no significant difference was present for both growth rate and yield (appended in Appendix 2 and Appendix 3). Recorded data from both controls were therefore pooled and used as a reference to determine growth rate and yield inhibition at the different test item concentrations.
Stimulation instead of inhibition of growth rate and yield was recorded at all test concentrations. No statistically significant differences were found between test item treatments and the pooled controls.

RANGE-FINDING TEST

The mean cell densities measured during the range-finding test are presented in Table 1 (below). Table 2 and Table 3 (below) present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

No significant inhibition of algal growth was found at any of the concentrations tested.

Based on these results, samples taken from the control and the two highest test concentrations were analysed. The concentrations measured at the start of the test were 0.00018 and 0.31 mg/L in the two highest test concentration, respectively, which decreased to 0.42-2.2% of the initial concentrations at the end of the test. Small responses were detected in the samples taken from the control, which were, however, most likely related to the analytical system since similar responses were seen in the analytical blanks (see also Table 2 of the appended Analytical Report - appendix 4).

Considering that the water solubility at a loading rate of 100 mg/L had been determined to be 2.5 µg/L, it appears likely that the highest test concentration had been oversaturated at the start of the test, which could have contributed to the concentration decrease during the exposure period.

Considering the results of the present range-finding test and the saturation test as well as the UVCB nature of the test item, it was decided to follow the recommendations described in the OECD series on testing and assessment number 23, 2019, and to adjust the procedure for preparing the test solutions by using stock solutions in acetonitrile to ensure reliable and more reproducible preparation of test solutions. To further account for the possibly hydrolytic instability of the test item, it was additionally decided to reduce the period for mixing test item and medium from 72 hours to 24 hours.

All test conditions were maintained within the limits prescribed by the study plan.

Table 1 - Mean Cell Densities (x10⁴ Cells/mL) during the Range-Finding Test

Time (h)	C894; WSF (mg/L)
	Control	1	10	100
0	1	1	1	1
72	201	200	209	189

Table 2 - Percentage Inhibition of Growth Rate during the Range-Finding Test

C894	Mean	Std. Dev.	n	%Inhibition
WSF (mg/L)
Control	1.767	0.0279	3
1	1.766	0.0107	3	0.005
10	1.781	0.011	3	-0.83
100	1.746	0.0259	3	1.2

Table 3 - Percentage Inhibition of Yield during the Range-Finding Test

C894	Mean	Std. Dev.	n	%Inhibition
WSF (mg/L)
Control	199.7	16.63	3
1	199.3	6.49	3	0.22
10	208.3	6.9	3	-4.3
100	187.6	14.36	3	6.1

FINAL TEST

Measured Test Item Concentrations

The results of analysis of the samples taken during the final test are described in Table 3 and Table 4 of the appended Analytical Report (appendix 4).

Samples taken from the stock solutions of C894 in ACN used in the preparation of test solutions were analysed. The analysed concentrations were at 63-82% relative to the nominal concentrations, indicating generally proper preparation of the stocks. Since the measured concentrations for the two highest stocks were below the level of nominal, the measured values were used for the further study evaluation following a worst-case assumption.

Small responses were detected in the samples taken from the control, which were, however, most likely related to the analytical system since similar responses were seen in the analytical blanks.

In addition, samples taken from all test concentrations and the controls were analysed. Small responses were observed at the retention time of the test item in most samples. These responses were below the response of the lowest calibration standards and could be estimated only by extrapolation of the calibration curve. Additionally, at the start of the test, the measured concentrations in the individual WAFs correlated with the applied dosing only at the two highest test concentrations.

Since the test item is a UVCB, the effect parameters were based on the analytically confirmed loading rates (see Table 4 below).

Table 4 - Nominal Loading Rate versus Confirmed Loading Rate

Stock			Test solution
Nominal conc.	Measured conc.	Applied volume per vessel	Final volume per vessel	Nominal loading rate	Confirmed loading rate
(µg/mL)	(µg/mL)	(mL)	(mL)	(µg/L)	(µg/L)
0.05	0.041	1	50	1	0.82
0.09	0.072	1	50	1.8	1.4
0.16	0.129	1	50	3.2	2.6
0.28	0.181	1	50	5.6	3.6
0.5	0.315	1	50	10	6.3

Mean Cell Densities

Figure 1 (attached graph) shows growth curves at different loading rates of C894. The individual and group mean cell densities measured at 24h intervals are given in Table 10 (in appendix 1).

Inhibition of Growth Rate and Inhibition of Yield

Table 5 (below) shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereas Table 6 (below) shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 7 (below) - see Appendix 1 (attached) for the individual values. Statistical analysis of the data is shown in Appendix 2 and Appendix 3 (attached).

Statistical comparison of algal growth between the blank and solvent control showed that no significant difference was present for both growth rate and yield (see Appendix 2 and Appendix 3 attached). Recorded data from both controls were therefore pooled and used as a reference to determine growth rate and yield inhibition at the different test item concentrations.

Stimulation instead of inhibition of growth rate and yield was recorded at all test concentrations. No statistically significant differences were found between test item treatments and the pooled controls.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Table 5 - Growth Rate and Percentage Inhibition for the Total Test Period

C894	Mean	Std. Dev.	n	%Inhibition
WAF(µg/L)
Pooled Control	1.982	0.0281	12
0.82	1.979	0.0063	3	0.11
1.4	1.992	0.038	3	-0.5
2.6	2.02	0.0188	3	-1.9
3.6	1.988	0.0202	3	-0.34
6.3	2.016	0.0324	3	-1.8

Table 6 - Growth Rate and Percentage Inhibition at Different Time Intervals

C894	n	0 – 24 h		24 – 48 h		48 – 72h
WAF(µg/L)		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Pooled Control	12	2.675		1.732		1.538
0.82	3	2.641	1.2	1.727	0.28	1.57	-2.1
1.4	3	2.62	2.1	1.789	-3.3	1.566	-1.8
2.6	3	2.678	-0.13	1.771	-2.3	1.61	-4.7
3.6	3	2.683	-0.32	1.722	0.58	1.56	-1.4
6.3	3	2.657	0.66	1.752	-1.2	1.64	-6.6

Table 7 - Yield and Percentage Inhibition for the Total Test Period

C894	Mean	Std. Dev.	n	%Inhibition
WAF(µg/L)
Pooled Control	382.055	31.4908	12
0.82	378.355	7.2295	3	0.97
1.4	394.085	43.5892	3	-3.1
2.6	427.53	24.1778	3	-12
3.6	389.078	23.3455	3	-1.8
6.3	424.071	41.49	3	-11

Determination of Effect Loading Rates

Table 8 shows the effect parameters based on analytically confirmed loading rates.

Table 8 - Effect Parameters

Parameter (µg/L)	NOELR	EL10	EL50
Growth rate	6.3	>6.3	>6.3
Yield	6.3	>6.3	>6.3

Experimental Conditions

Table 9 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 units).

During the exposure period the temperature measured in the incubator was maintained between 22 and 24°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).

Table 9 - pH Levels Recorded during the Final Test

C894	pH
WAF(µg/L)	t=0h	t=72h
Blank control	8	7.6
Solvent control	8	7.7
0.82	8	7.7
1.4	8	7.6
2.6	8	7.6
3.6	8	7.6
6.3	8	7.6

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of the test item tested.

The 72h-EL50 for growth rate inhibition (ERL50) and yield inhibition (EYL50) exceeded a loading rate of 6.3 µg/L.
The 72h-NOELR for both growth rate inhibition and yield inhibition was 6.3 µg/L.

Due to the very low solubility of the test item in water, concentration levels that might be toxic for algae could not be reached.

Executive summary:

The objective of the study was to evaluate the test item for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.

A full test was performed based on the results of a range-finding test. The study met the acceptability criteria prescribed by the study plan and was considered valid. Test conditions and results of the final test are presented below:

Test Item:	2,2-bis[({3-[methyl(phenyl)amino]propanoyl}oxy)methyl]butyl 3-[methyl(phenyl)amino]propanoate
Appearance	Light orange liquid
Purity	UVCB
Preparation of test solutions	WAFs individually prepared at nominal loading rates in the range of 1.0 to 10 mg/L, using stock solutions in acetonotrile.
Specific procedures	Glassware was wrapped in aluminum foil and preparation of test solutions was performed under dimmed light conditions to minimize exposure to light.
Experimental Set-up
Guidelines	OECD TG 201 and OECD GD 23
Test concentrations	WAFs individually prepared at a loading of 1.0, 1.8, 3.2, 5.6 and 10 µg/L
Controls	Blank control and solvent control
Stock solutions	0.050 - 0.50 µg/mL in acetonitrile
Number of replicates	6 replicates per control group and 3 per test concentration
Sampling for analysis	Stock solutions at t=-48h.
	Test solutions at t=0h, t=24h, and t=72h.
Experimental conditions
pH and temperature	Within the ranges specified in OECD TG 201
Light regime	Continuous
Results
Actual exposure concentrations	0.82, 1.4, 2.6, 3.6 and 6.3 µg/L (analytically confirmed loading rates)
Recorded effects	Statistical comparison of algal growth between the blank and solvent control showed that no significant difference was present for both growth rate and yield. Recorded data from both controls were therefore pooled and used to determine growth rate and yield inhibition at the different test item concentrations.
	Stimulation instead of inhibition of growth rate and yield was recorded at all test concentrations. No statistically significant differences were found between test item treatments and the pooled controls.

The effect parameters (based on analytically confirmed loading rates) obtained in this study are summarized in the table below.

Parameter (µg/L)	NOELR	EL10	EL50
Growth rate	6.3	>6.3	>6.3
Yield	6.3	>6.3	>6.3

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.3 µg/L

EC10 or NOEC for freshwater algae:

6.3 µg/L

Additional information

Growth rate (µg/L) NOELR 6.3; EL10 >6.3; EL50 >6.3 and
Yield (µg/L) NOELR 6.3; EL10 >6.3; EL50 >6.3