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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

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:

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:

Concentrations: During the final test, samples for possible analysis were taken from all test concentrations. Stock solutions in ACN used for preparation of test solutions were sampled as well.

Sampling method:
Frequency 1. At the start of the test and after 24 hours from the freshly prepared solutions.
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
Volume 1.0 mL from the approximate centre of the test vessels

Sample storage conditions before analysis:
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 each refreshment period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not 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

Method:
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.

Combined limit/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.

Full tests
Based on the results obtained in the combined limit/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.


Differential loading: WSF prepared at a loading rate of 100 mg/L
Controls: Solvent contol - 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).
Chemical name of vehicle: Acetonitrile
Concentration of vehicle in test medium: 1mL acetonitrile : 50 mL test medium (~2%)
Evidence of undissolved material: All test solutions were clear and colourless at the end of the preparation procedure.
Other relevant information: 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.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Age at study initiation: Young daphnids with an age of < 24 hours, from.
Feeding during test: No feeding


Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Source: In-house laboratory culture with a known history.
Age of parental stock: Parental daphnids of more than two weeks old
Feeding during breeding: Daily, a suspension of fresh water algae.
Maximum age of cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Culture medium: M7, as described by OECD 211 TG.

The following salts and vitamins were added to freshly prepared test medium (see section 4.9.3.2) to reach the following concentrations:
Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L


ACCLIMATION
- Acclimation period: No details on acclimation reoprted, daphnids <24 h old at test initiation

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Remarks on exposure duration:

According to OECD guideline

Post exposure observation period:

No post exposure observation period

Hardness:

180 mg/L - expressed as CaCO3

Test temperature:

21 - 22 °C

pH:

7.9 - 8.2

Dissolved oxygen:

≥ 8.9 mg/L at the end of the test

Salinity:

Not reported - freshwater study

Conductivity:

Not reported - freshwater study

Nominal and measured concentrations:

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

Nominal conc. of stock solutions (µg/mL): 0.050, 0.090, 0.16, 0.28, 0.50
Average measured conc. of stocke solutions(µg/mL): 0.056, 0.10, 0.17, 0.31, 0.52

Details on test conditions:

TEST SYSTEM
Test vessel: 100 ml, all-glass
Volume of solution: 50 mL
Aeration: No aeration of the test solutions
Renewal rate of test solution: Renewal of test solutions after 24 hours
No. of organisms per vessel: 5
No. of vessels per concentration: 4
No. of vessels per control: 4 blank control
No. of vessels per vehicle control: 4 solvent control

TEST MEDIUM / WATER PARAMETERS
The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L


OTHER TEST CONDITIONS
Photoperiod: 24 hour darkness

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a WSF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:

- Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WSFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test.
- Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.
- Glass test vessels of 60 mL were used.
- The test was performed statically without renewal of test solutions.
- Samples were taken at the start and end of the test and were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Saturation Test
This test was intended to determine the time of mixing required to reach the maximum concentration soluble in test medium under the given test conditions, defined as the saturation concentration, and the preparation conditions that are required to achieve it.
A loading rate of 100 mg/L was prepared and continuously stirred for three days. Approximately every 24 hours, the mixture was allowed to stabilize for two hours before a sample was taken for possible chemical analysis. Since the test item was a liquid, subsamples of approximately 10 mL were collected by siphoning before the sample for chemical analysis was taken.
The samples were stored under the same conditions, and analysed together, with the samples taken during the simultaneously performed combined limit/range-finding test.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 10 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Details on results:

Immobility
No immobility was observed in the blank control and at any of the test concentrations throughout the exposure period. In the solvent control, two and one daphnids were observed to be immobile after 24 and 40 hours of exposure, respectively. It should be noted that ≤10% immobility is accepted in the control, and therefore effects below 10% were considered biologically not relevant. Table 3 (See 'any other information on results incl. tables' section below) shows the responses recorded during the final test.
The responses recorded in this test allowed for reliable determination of an EL50.

Combined Limit/Range-Finding Test

Table 1 (below) shows the responses recorded during the range-finding test. No immobility was observed in the control or at any of the test concentrations during the test period.

Based on these results, samples taken from the control and the highest test concentration were analysed. The concentration measured at the start of the test was 0.0035 mg/L in the highest test concentration, which decreased to 10% of the initial concentration 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 or higher responses were seen in the analytical blanks (see also Table 3 of the appended Analytical Report).

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.

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

Table 1 - Incidence of Immobility in the Combined Limit/Range-Finding Test

Time (h)	Replicate	C894; Loading rate (mg/L)
		Control	1	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
Total introduced		20	10	10	20
24	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
Total immobilised		0	0	0	0
Effect %		0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
Total immobilised		0	0	0	0
Effect %		0	0	0	0

Saturation Test

The results of analysis of the samples taken during the saturation test are described in Table 2 of the appended Analytical Report.

The concentration measured in the solution prepared at a loading rate of 100 mg/L and constantly stirred until two hours before sampling was 0.80 mg/L after 24 hours of agitation and 0.59 and 0.38 mg/L after 48 and 72 hours of agitation, respectively. Based on these results it was concluded that the maximum saturation of the test item in test medium was reached after 24 hours of agitation. Furthermore, the decline of concentrations over time is likely related to hydrolytic instability of the test item as an additional factor explaining the decrease of measured concentrations.

Considering the results of the combined limit/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, and to perform a full test with renewal of test solutions after 24 hours of exposure.

Final Test

Measured Test Item Concentrations

The results of analysis of the samples taken during the final test are described in Table 4 and Table 5 of the appended Analytical Report.

Samples taken from the stock solutions of the test item in ACN used in the preparation of test solutions were analysed. The analysed concentrations for the first renewal period were at 88-97% relative to the nominal concentrations and for the second renewal period at 121-133% relative to the nominal concentrations, indicating generally proper preparation of the stocks. All calculated average exposure concentrations were in agreement with the nominal concentrations (89-96%, see Table 2 below). 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 or end of each renewal period, the measured concentrations in the individual WAFs did not correlate with the applied dosing.

Since the test item is a UVCB, the effect parameters were based on the applied nominal loading rates.

Table 2 - Average Concentration Versus Nominal Concentration of Stock Solutions

Test item	Measured conc. (mg/L)		Average	Relative to nominal
Nominal conc. (µg/mL)	t=0d	t=32d	(µg/mL)	[%]
0.05	0.045	0.0664	0.056	90
0.09	0.087	0.115	0.1	89
0.16	0.149	0.197	0.17	92
0.28	0.272	0.341	0.31	91
0.5	0.44	0.605	0.52	96

Table 3 - Incidence of Immobility in the Final Test

Time (h)	Replicate	Test item; WAF (µg/L)
		Blank	Solvent Control	1	1.8	3.2	5.6	10
		Control
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
Total introduced		20	20	20	20	20	20	20
24	A	0	0	0	0	0	0	0
	B	0	0	0	0	0	0	0
	C	0	0	0	0	0	0	0
	D	0	2	0	0	0	0	0
Total immobilised		0	2	0	0	0	0	0
Effect %		0	10	0	0	0	0	0
48	A	0	0	0	0	0	0	0
	B	0	0	0	0	0	0	0
	C	0	0	0	0	0	0	0
	D	0	1	0	0	0	0	0
Total immobilised		0	1	0	0	0	0	0
Effect %		0	5	0	0	0	0	0

Determination of Effect Concentrations

Table 4 shows the effect parameters based on nominal loading rates.

Table 4 - Effect Parameters

Parameter	Test Item; WAF (µg/L)
24h-, 48h-EL50	>10

Experimental Conditions

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 5. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test). The temperature of the test medium varied between 20 and 21°C when measured in the fresh and old solutions upon renewal of the test solutions after 24 hours of exposure. The temperature continuously measured in a temperature control vessel varied between 21 and 22°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Table 5 - pH and Oxygen Concentrations (mg/L) Measured during the Final Test

Test tem	t=0h (fresh)		t=24h (old)		t=24h (fresh)		t=48h (old)
WAF (µg/L)	pH	O2	pH	O2	pH	O2	pH	O2
Blank control	8.2	9.1	7.9	9.3	7.9	9.3	8.0	8.9
Solvent control	8.2	9.1	7.9	9.4	8.0	9.3	8.0	9.0
1	8.2	9.2	7.9	9.4	8.0	9.3	8.0	9.0
1.9	8.2	9.1	7.9	9.4	7.9	9.3	8.0	9.0
3.2	8.1	9.2	7.9	9.4	7.9	9.4	8.0	9.0
5.6	8.1	9.1	7.9	9.4	7.9	9.4	8.0	9.0
10	8.2	9.1	7.9	9.4	7.9	9.4	8.0	9.0

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EL50 for Daphnia magna exposed to the test item was beyond the range of concentrations tested, i.e. exceeded a nominal loading rate of 10 µg/L.

Executive summary:

The objective of the study was to evaluate the test item for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EL50 at 24 and 48 hours of exposure.

A full test was performed based on the results of a combined limit/range-finding test and a saturation 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 in the table 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 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 202 and OECD GD 23
Exposure design	Semi-static with renewal after 24 hours
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	4 replicates containing 5 daphnids each
Sampling for analysis	Stock solutions at t=-48h and t=-24h.
	Freshly prepared test solutions (t=0 and 24 h) and 24-hour old test solutions (t=24 and 48 h)
Experimental conditions
pH, Temperature, Dissolved Oxygen	Within the ranges specified in OECD TG 202
Light regime	The study was performed in the dark due to the light sensitive nature of the test item
Results
Actual exposure concentrations	1.0, 1.8, 3.2, 5.6 and 10 µg/L (analytically confirmed nominal loading rates)
Recorded effects	No immobility was observed in the blank control and at any of the test concentrations throughout the exposure period. In the solvent control, two and one daphnids were observed to be immobile after 24 and 40 hours of exposure, respectively. It should be noted that ≤10% immobility is accepted in the control, and therefore effects below 10% were considered biologically not relevant.

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

Parameter	Test Item; WAF (µg/L)
24h-, 48h-EL50	>10

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 µg/L

Additional information

24 & 48 h EL50 (water accomodated fraction) : >10 µg/L