Reactionmass of octan-2-yl prop-2-enoate and octan-3-yl prop-2-enoate and octan-4-yl prop-2-enoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

19th - 22nd November, 2019

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M FMRD, S352546
- Expiration date: 2020.05.01
- Purity: 99.7%
- Physical state: Clear and colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature (15-25°C).
- Stability under test conditions: Due to analytical issues, stability during the test is unknown.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.146, 0.367, 0.921, 2.31, 5.81, 14.6 mg/L and blank control
- Sampling method: For determination of test item concentrations in the static test, samples were taken from test group in 0 hour fresh solutions and 24, 48 and 72 hour old solution. The residual initial solutions were used to measure initial concentrations (15mL sample from test and control solutions). For sampling at 24, 48 and 72 hours, 5mL solutions was sampled from each replicate in each test and control group and pooled. 5mL of each sample was pre-treated for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared by dilution of a stock solutions due to the limitations of weighing a small amount of the test item. A test solution at a nominal concentration of 14.6 mg/L prepared with OECD alga growth medium. 0.01460g of the test item was weighted and 1000 mL growth medium was added to dissolve the test item. The solution was mixed by magnetic stirring at 350 rpm for 23 hours and 40 mins. The saturated solutions was then allowed to stand in a separating funnel for 34 minutes and the lower solution was runoff and collected in a flask. 5.00, 12.6, 31.5, 79.1, and 199 mL of the collected solution was pipetted into the 500 mL of the OECD alga growth medium to make nominal test concentrations of 0.156, 0.367, 0.921, 2.31 and 5.81 mg/L. These solutions were mixed thoroughly. 500mL of the undiluted solution was used as the 14.6 mg/L solution.
- Controls: Test medium without test substance or other additives.
- Test concentration separation factor: 2.5x
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source: Stock from Institute of Hydrobiology, Chinese Academy of Sciences, propagated in house.
- Age of inoculum: Four days
- Method of cultivation: propagation under same conditions as test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.1-23.9 °C

pH:

7.92-8.20

Nominal and measured concentrations:

Nominal concentrations: 0.146, 0.367, 0.921, 2.31, 5.81, 14.6 mg/L
Measured initial concentrations: 0.0026, 0.0471, 0.166, 1.31, 4.35 and 12.1 mg/L
Geometric mean concentrations old media: 0.0009, 0.0012, 0.0015, 0.0021, 0.0016 and 0.0064 mg/L
Geometric mean concentrations (overall): 0.0012, 0.0029, 0.0048, 0.0105, 0.0113 and 0.0423 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL, glass conical flasks, 100 mL fill volume. Capped with air permeable membrane.
- Aeration: Shaking at 100±20 PRM
- Renewal rate of test solution: None
- Initial cells density: 5000 cell/mL
- Control end cells density: 294000 cell/mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): three

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4650-4730 Lux, cool white light
- Other: pH measured at beginning and end of test, light intensity measured daily with five points at test vessel level, temperature continuously monitored.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Manual cell counting by improced Neubauer type with microscope daily
- Other: abnormal cells also determined daily by microscopy

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0, 0.146, 1.46, 14.6 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.042 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 14.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No abnormal appearance in controls or exposed algae
- Any stimulation of growth found in any treatment: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

72hr ErC50=2.35 (95% CI: 1.79-3.08). Test run on 5th-8th July 2019 (134 days before definitive test)

Table 2. Growth Inhibition of Test Item to Alga in the Definitive Test

Nominal conc (mg/L)	Replicate	Mean Cell conc (x10^4 cell/mL): 0d	Mean Cell conc (x10^4 cell/mL): 1d	Mean Cell conc (x10^4 cell/mL): 2d	Mean Cell conc (x10^4 cell/mL): 3d	Mean Spec Growth Rate: 0-1d	Mean Spec Growth Rate: 1-2d	Mean Spec Growth Rate: 2-3d	Mean Spec Growth Rate: 0-3d	CV of Daily growth rate	Inhibition (%)	p for rate (0.05, 2 side)
Control	1	0.5	1.8	6.5	29.8	1.28	1.28	1.52	1.36	10.2%
	2	0.5	1.5	6.3	29.5	1.10	1.44	1.54	1.36	17.0%
	3	0.5	1.8	6.8	28.8	1.28	1.33	1.44	1.35	6.1%
	Mean	/	1.7	6.5	29.4	1.22	1.35	1.50	1.36	11.1%
	CV%	/	10.2	3.9	1.7	8.5	6.1	3.5	0.4	/	/	/
0.146	1	0.5	1.5	7.0	28.5	1.10	1.54	1.40	1.35	16.7%
	2	0.5	1.5	6.5	28.0	1.10	1.47	1.46	1.34	15.7%
	3	0.5	1.8	6.8	30.3	1.28	1.33	1.49	1.37	8.0%
	Mean	/	1.6	6.8	28.9	1.16	1.45	1.45	1.35	13.5%
	CV%	/	10.8	3.7	4.2	9.0	7.4	3.2	1.1	/	0.7	0.74
0.367	1	0.5	1.5	6.8	29.8	1.10	1.51	1.48	1.36	16.8%
	2	0.5	1.8	7.0	29.0	1.28	1.36	1.42	1.35	5.2%
	3	0.5	1.3	6.0	30.0	0.96	1.53	1.61	1.36	25.9%
	Mean	/	1.5	6.6	29.6	1.11	1.47	1.5	1.36	16.0%
	CV%	/	16.8	8.0	1.80	14.5	6.3	6.5	0.4	/	0.0	1.00
0.921	1	0.5	1.8	6.8	28.5	1.28	1.33	1.43	1.35	5.7%
	2	0.5	2.0	7.5	31.3	1.39	1.32	1.43	1.38	4.0%
	3	0.5	1.8	7.0	28.8	1.28	1.36	1.41	1.35	4.9%
	Mean	/	1.9	7.1	29.5	1.32	1.34	1.42	1.36	4.9%
	CV%	/	6.1	5.1	5.2	4.8	1.6	0.8	1.30	/	0.0	0.77
2.31	1	0.5	1.8	7.0	28.3	1.28	1.36	1.40	1.35	4.5%
	2	0.5	1.8	6.5	29.3	1.28	1.28	1.51	1.36	9.8%
	3	0.5	1.5	6.3	29.5	1.10	1.44	1.54	1.36	17.0%
	Mean	/	1.7	6.6	29.0	1.22	1.36	1.48	1.36	10.4%
	CV%	/	10.2	5.5	2.2	8.5	5.9	5.0	0.4	/	0.0	1.00
5.81	1	0.5	1.5	6.8	30.0	1.10	1.51	1.48	1.36	16.8%
	2	0.5	1.8	7.3	29.3	1.28	1.40	1.39	1.36	4.9%
	3	0.5	1.8	6.8	29.5	1.28	1.33	1.47	1.36	7.2%
	Mean	/	1.7	7.0	29.6	1.22	1.41	1.45	1.36	9.6%
	CV%	/	10.2	4.1	1.2	8.5	6.4	3.4	0.00	/	0.0	0.37
14.6	1	0.5	1.8	7.0	27.3	1.28	1.36	1.36	1.33	3.5%
	2	0.5	1.8	7.0	27.8	1.28	1.36	1.38	1.34	3.9%
	3	0.5	1.5	6.5	28.8	1.10	1.47	1.49	1.35	16.2%
	Mean	/	1.7	6.8	28.0	1.22	1.40	1.41	1.34	7.9%
	CV%	/	10.2	4.2	2.7	8.5	4.5	5.0	0.7	/	1.5	0.07

Note: 1. Inhibition was precent inhibition in average specific growth rate. 2. CV is the coefficient of variation in replicate culture., 3. /: means the value did not need to be calculated.

Validity criteria fulfilled:

yes

Remarks:

control biomass increase >16 (57.8x), CV for section-by-section growth rates ≤35% (11.1%), CV for average growth rates <7 (0.4%).

Conclusions:

72 hour EC50 > 14.6 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
72 hour EC50 > 0.043 mg/L (geometric mean) in Pseudokichneriella subcapitata (OECD 201).

72 hour NOEC =14.6 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
72 hour NOEC = 0.043 mg/L (geometric mean) in Pseudokichneriella subcapitata (OECD 201).

Executive summary:

The 72 hour EC50 to Pseudokichneriella subcapitata was determined in a  test according to OECD 201 guidelines. Nominal concentrations of 0.146, 0.367, 0.921, 2.31, 5.81, 14.6 mg/L and blank control were run using 3 replicates per concentration and no dispersant.  The measured test concentrations were close to nominal in fresh solutions,  could not be reliable detected in old solutions after 24 hours.   Measured geometric mean concentrations of the substance during the test were 0.0012, 0.0029, 0.0048, 0.0105, 0.0113 and 0.0423 mg/L. No impairment of growth rate nor abnormal cells were observed throughout the test when compared to the control. An EC50 >14.6 mg/L (nominal concentration) and EC50 > 0.043 mg/L measured) were determined. A NOEC of >14.6 mg/L (nominal concentrations) and NOEC > 0.043 mg/L (measured) were also determined.

This study was conducted under international guidelines and was compliant with GLP criteria. However, the analytical data for the test indicates that the effects concentration values reported in this study are not reliable. From available data in the study report, the analytical data does not confirm test substance concentration and shows the test substance was not maintained throughout the test.  The mechanisms of the chemicals disappearance is not understood and was unexpected given results of an analogous compound. Further, no toxicity to algae was found for test solutions, which is contradictory to results for the analogous substance. For these reasons, this study has been deemed unreliable. Thus, these results are not considered suitable for use in Risk Assessment, Classification & Labeling, and PBT analysis.

.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

21 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE : Formulation encoded in Microsoft Excel

2. MODEL: Linear regression of acrylate ester 72-hour toxicity to Pseudokirchneriella subcapitata

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: C8H170C(=O)C=C. Model depends on carbon number in alkyl chain.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Short-term toxicity to algae (inhibition of the exponential growth rate)
- Unambiguous algorithm: linear regression QSAR
QSAR model based on a single descriptor. Log (1/EC50) = 0.1536 * Carbon number - 1.6978
- Defined domain of applicability:
The QSAR model was developed specifically for acrylate esters of middle molecular weight having approximately eight carbons in its alcoholic domain (i.e., carbon number = 8). All training set chemicals contained the same functional groups, an acrylic acid esterified with a saturated aliphatic alcohol, and differed only by number of carbons in the alcoholic moiety. USEPA indicates that high molecular weight acrylates having log P > 5 are toxic by simple narcosis, but display excess toxicity at lower log P values. The mechanism of toxicity of small acrylate esters is by protein adduct formation via Michael-type addition. The model is suitable only for estimation of toxicity of simple acrylate esters showing toxicity by a Michael addition mechanism. Complex esters such as bifunctional acrylates or esters of diol or hydroxylated amines, as well as high molecular weight acrylate esters are excluded from the applicability domain of this model.
- Appropriate measures of goodness-of-fit and robustness and predictivity:
The model was trained with four chemicals:
Predictor: Slope:0.1536 (Standard error:0.0123)
Predictor: Intercept:-1.6978 (Standard error:0.0417)
Correlation coefficient (R-squared): 0.987
Fisher's test (F-statistic): 155.99
Standard error of the estimate: 0.0389
The model was evaluated by leave-one-out internal cross-validation
Q2LOO = 0.9993 (explained variance in prediction)
- Mechanistic interpretation: The toxicity of low molecular weight acrylate esters is by protein adduct formation via a Michael-type addition mechanism. All member of the training set are expected to follow this pathway, as demonstrated by the quality of the regression. The use of carbon number, which is an unambiguous value inherent to the chemicals, is a surrogate for other qualities such as hydrophobicity or molecular weight. Toxicity increases on a molar concentration basis with molecular weight (and concomitantly, hydrophobicity) due to increased ability of the molecule to reach its active site. The excess toxicity is mitigated in high molecular weight acrylate esters with logP>5.

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain: The Carbon number of the simulated chemicals is beyond the range of the training set. However, acrylate esters as described in the QMRF may have two modes of toxicity. High molecular weight acrylate esters are toxic by a simple narcotic mechanism. Low molecular weight acrylates show excess toxicity by a mechanism involving Michael-type addition. Toxicity measurements for the related UVCB substance IOA (having an average Carbon number of 8) in Pseudokirchneriella subcapitata demonstrate that isooctyl acrylate also shows excess toxicity, and therefore is better predicted by this model than by a mechanism of simple narcosis. Toxicity of MTDID 44428 is expected to be similar.
- Structural domain: The training set chemicals are simple esters containing acrylate and either linear or branched aliphatic alcohols as the sole functional groups. Isooctyl acrylate is within the structural domain of the model.
- Mechanistic domain: Acrylate esters as described in the QMRF may have two modes of toxicity. High molecular weight acrylate esters are toxic by a simple narcotic mechanism. Low molecular weight acrylates show excess toxicity by a mechanism involving Michael-type addition. Initial toxicity measurements in Pseudokirchneriella subcapitata demonstrate that the related UVCB substance IOA also shows excess toxicity, and follows the Michael-type addition mechanism of the training set chemicals. It should be noted that this is an inherently conservative model, in that toxicity will be overestimated to the degree that an acrylate acts through a narcotic, rather than Michael addition, mechanism.
- Similarity with analogues in the training set: The training set for the QSAR consists entirely of structural analogues, i.e., simple esters containing one branched or linear aliphatic alcohol esterified with acrylic acid.

6. ADEQUACY OF THE RESULT
The modeled result fulfills the need for a valid test of toxicity to algae. The model was based entirely on experimental data for structural analogs. The toxicity data used to derive this model was obtained from reliable sources according to a consistent, internationally-accepted protocol (i.e., OECD TG201). The descriptor, Carbon number, is unambiguous and not subject to experimental error. Despite limited data availability for toxicity of simple acrylate esters, the resulting model was highly linear. The inclusion of a branched acrylate ester, 2-methylbutyl acrylate, taken with the linearity of the result, demonstrates that toxicity of acrylate esters has little dependence on branching of the esterified alcohol subunit. The variance of model predictions was small. Therefore, this study is classified as acceptable as a QSAR simulation of toxicity to algae. Its use in evaluating aquatic toxicity is considered adequate for regulatory purposes. The results are used in risk assessment and PBT analysis.

Guideline:

other: REACH guidance on QSARs R.6, May/July 2008

Principles of method if other than guideline:

Linear regression of acrylate ester 72-hour toxicity to Pseudokirchneriella subcapitata. All studies from which data were obtained were conducted under OECD TG201.

GLP compliance:

not specified

Test organisms (species):

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.542 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.289 - 1.03 mg/L

Table 1, Analogous compound data for quantitative read-across
Name	CAS#	MW	Carbon number	Log P			P. subcapitataEC50 (mg/L)
				Value	Method:	Source	Value	Source
n-Methyl acrylate	96-33-3	86.1	1	0.739	OECD TG107	IUCLID^1	3.1	NITE^4
n-Ethyl acrylate	140-88-5	100.1	2	1.32	Experimental	SRC^2	2.3	NITE
n-Butyl acrylate	141-32-2	128.2	4	2.36	Experimental	SRC	1.7	NITE
2-Methylbutyl acrylate	44914-03-6	142.2	5	2.62	Simulated	KOWWIN^3	1.15	Sponsor data
2-Ethylhexyl acrylate	103-11-7	184.2	8	4.64	OECD TG107	IUCLID	1.71^5	ECB^6
1. IUCLID Data set, European Chemicals Bureau
2. SRC PhysProp Database, Syracuse Research Corp.
3. KOWWIN v. 1.67, Syracuse Research Corp.
4. NITE, data from Japan National Institute of Technology and Evaluation, following method OECD201
5. 72-Hour EC50 forDesmodesmus subspicatus, following method OECD201
6. Cited in ECB Risk Assessment Report for 2-ethylhexyl acrylate:

Table 2, Summary of statistics from 'leave-one-out' analysis
Submodel excluding:	Slope	Intercept	Predicted toxicity
			log (1 / EC50)	EC50 (mg/L)
n-Methyl acrylate	0.1466	-1.6682	-0.4952	0.576
n-Ethyl acrylate	0.1581	-1.7225	-0.4578	0.529
n-Butyl acrylate	0.1596	-1.7008	-0.4239	0.489
2-Methylbutyl acrylate	0.1410	-1.6757	-0.5478	0.650
Average of submodel predictions	0.1513	-1.6918	-0.4812	0.561
Standard deviation	0.0090	0.0248	0.0531	0.069
Prediction using average regression coefficients of submodels	─	─	-0.4814	0.558
Full model	0.1536	-1.6978	-0.4690	0.542
Standard error	0.0123	0.0417	0.0645	─

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EC50 of MTDID 44428 is 0.542 mg/L based on quantitative read-across using analogous compound data.

Executive summary:

The 72-hour EC50 of MTDID 44428 to the green algae Pseudokirchneriella subcapitata was assessed using a QSAR based on analogous compound data. Data were collected from accepted databases for toxicity and log P of three acrylate to Ps. subcapitata (n-methyl acrylate, n-ethyl acrylate, and n-butyl acrylate). A fourth compound, 2-methylbutyl acrylate, was additionally available as a sponsor-owned ecotoxicology study. Experimentally determined logP was not available for 2-methylbutyl acrylate, and a simulated value was used instead. EC50 values were converted to micromolar concentration units and transformed as log (1/EC50). Transformed data was entered into linear regression models using either logP or number of carbon atoms in the esterified alcohol subunit. Regression using the carbon number gave the best performance. The regression equation was log (1/EC50) = 0.1536 * Carbon number - 1.6978, R2 = 0.9873, P = 0.003. The 'leave-one-out' cross-validation statistic was 0.9993. The predicted EC50 was 0.542 mg/L.

The model training set consists of acceptable data from chemicals with the same reactive groups. The regression coefficient and cross-validation statistic had high values. Therefore, the model is valid and the test substance is within its applicability domain.

Results Synopsis

Test Type: static (quantitative read-across based on data obtained using OECD201 methodology)

EC50: 0.542 mg/L                     95% C.I.: 0.289 - 1.03 mg/L

NOEC: Not determined              Probit Slope: not applicable

Description of key information

72 hour EC50=0.542 mg/L in Pseudokirchneriella subcapitata based on quantitative read-across using analogous compound data.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.542 mg/L

Additional information

A study performed using MTDID 44428 with Pseudokirchneriella subcapitata under OECD 201 was considered unreliable due to methodological deficiences. The data gap for toxicity to aquatic algae and cyanobacteria was filled by using a QSAR specifically developed for middle weight acrylate esters. The model training set consists of acceptable data from chemicals with the same reactive groups. The regression coefficient and cross-validation statistic had high values. Therefore, the model is valid and the test substance is within its applicability domain. The 72 hour EC50 was thus calculated to be 0.542 mg/L.