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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To achieve the nominal test concentrations 1.00, 2.15, 4.64, 10.00, 21.50 and 46.40 g of test substance were added to the test water using an ultra-turrax stirrer, respectively. A total of 25 minutes were needed from the beginning of test solution preparations until their termination. Once all solutions were ready the fish were subsequently placed then placed into the aquaria. (For all concentrations the fish were placed in the unfiltered test solution, the test solution was only filtered before analysis)
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Length at study initiation (length definition, mean, range and SD): 3.25 cm (2.9 - 3.8 cm)
- Weight at study initiation (mean and range, SD): 0.21 g (0.13 - 0.37 g)
- Corpulence factor of the batch: 0.6
- Feeding during test: Withdrawal of food: 1 day before and during exposure
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Nominal and measured concentrations:
Nominal test concentrations (mg/l):
0, 100, 215, 464, 1000, 2150, 4640
Details on test conditions:
TEST SYSTEM
- Fill volume: 10 l
- Aeration: None
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.2 g fish / l water


OTHER TEST CONDITIONS
- Photoperiod: Photoperiod: 16 : 8 hours day-night regime
-

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Results used to determine the conditions for the definitive study: LC 50 after 96 h: between 1000 and 10000 mg/l
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
271.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
other: LC20
Effect conc.:
604.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 216.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1 422.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Reported statistics and error estimates:
The LC50 was statistically evaluated from the LC20 value (604.5 mg/l) and the LC100 value (1422.3 mg/l).
LC50 (1 h): > 4640* mg/l
LC50 (4 h): ca. 4640* mg/l
LC50 (24 h): 3474 (2476 - 5906)* mg/l
LC50 (48, 72, 96 h): > 2150 < 4640* mg/l

2 and 10 fish exposed to 2150* and 4640* mg test substance/l died respectively. No deaths were observed at 1000* mg/l and less. Symptoms of toxicity (gasping and slight apathy) were limited to the highest dose groups.
(* = nominal concentration)

LC50 after 1 and 96 hours
(Based on the mean analytically detected concentrations, unfiltered samples)

Analytical / No of / dead / /
conc. / fish / fish / mort. /
(mg/l) / Start / after 96 h / % /
--------------------------------------------
16.8 / 10 / 0 / 0.0 /
33.8 / 10 / 0 / 0.0 /
101.5 / 10 / 0 / 0.0 /
271.3 / 10 / 0 / 0.0 /
604.5 / 10 / 2 / 20.0 * /
1422.3 / 10 / 10 / 100.0 * /

LC50 > 604.5 (10 % significance level)
LC50 < 1422.3 (1 % significance level)

ANALYTICAL MONITORING:
The analytically detected concentrations of the unfiltered samples were initially in the range of 29.5 % - 59.5 % and after about 96 h between 1.2 - 4.4 % of the nominal concentrations.
The analytically detected concentrations of the filtered samples were initially in the range of 0.6 - 2.0 % and after about 96 hours between 0.0 - 1.0 % of the nominal concentrations.

OBSERVATIONS:
All test solutions showed a certain level of turbidity (milky in colour) and fat-like drops were visible on the water surface, both increasing with each increase in the concentration of the test substance respectively.

Due to the high level of turbidity within the two highest concentrations (2150 and 4640 mg/l) it was necessary to direct the fish the front pane of the aquaria using a net so that observations for fish mortalities or for possible symptoms of toxicity could be made.
Conclusions:
In the acute toxicity test to Brachydario rerio with the tested substance a LC50 of 1,216.1 mg/L and an LC100 of 1,422.3 mg/L (96h) was
determined. The test was performed according to OECD guideline no. 203.
Executive summary:

The short-term toxicity of the test substance to Brachydanio rerio was tested according to OECD guideline no. 203. The NOEC was 271.3 mg/L, whereas the minimal concentration leading to 100% mortality in the tested species was determined at 1,422.3 mg/L (=LC100). The LC50 (1,261.1 mg/L) was mathematically derived from the LC20 value (604.5 mg/L) and the LC100 value (1,422.3 mg/L).

The target substance and the tested substance are both esters of p-methoxycinnamic acid. Due to this close structural relationship, these two compounds are considered to show comparable ecotoxicological properties. Therefore, the present results are used in a read-across approach to support the findings made in the ready biodegradation study conducted with isopentyl p-methoxycinnamate.

 

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

Scenario 2 of the Read Across Assessment Framework (RAAF) is appropriate because it is hypothesised that the target and source substances present similar physico-chemical, environmental and toxicological properties, as a result of their structural similarity (i.e. analogue approach). The source substance 2-ethylhexyl 4-methoxycinnamate (OMC) contains the same functional groups as Isopentyl p-methoxycinnamate (IMC), differing only in the length of the carbon chain originating from the alcohol.

Physico-chemical properties of the two substances are broadly similar. Both substances are liquids with low melting / freezing points. Both Isopentyl p-methoxycinnamate and 2-Ethylhexyl 4-methoxycinnamate are poorly soluble, with high partition coefficients and low vapour pressure. Relative density values of the two substances are within a narrow range of 1.0104 to 1.04. Boiling point, flash point and auto-ignition temperatures of the two substances are also similar.

Due to the similarity in chemical structure and physico-chemical properties, both substances are expected to behave in a similar manner when released into the environment and the source and target substance are believed to present comparable bioavailability and behave comparably in biological systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source: 2-ethylhexyl 4-methoxycinnamate
Target: Isopentyl p-methoxycinnamate

The target substance, Isopentyl p-methoxycinnamate is an organic mono-constituent substance used as a sunscreen agent in cosmetics. The target substance has a typical degree of purity of ≥ 96.5% ≤ 99.93% w/w. Impurities include 3-Methylbutyl (Z)-p-methoxycinnamate (≥ 0.01% ≤ 0.5% w/w); 3-Methylbutyl p-methoxy-dihydrocinnamate (≥ 0.01% ≤ 0.5% w/w); 3-Methylbutyl 3-isopropoxy-3-(p-methoxyphenyl)propionate (≥ 0.01% ≤ 0.5% w/w); 4,4’-Dimethoxystilbene (≥ 0.01% ≤ 0.5% w/w); Pentyl (E)-p-methoxycinnamate (≥ 0.01% ≤ 0.5% w/w); 3-Methylbutyl-3-methyl-p-methoxycinnamate (≥ 0.01% ≤ 0.5% w/w); 3-Methylbutyl p-methoxy-3-(3-methylbutoxy)cinnamate (≥ 0.01% ≤ 0.5% w/w). None of the impurities are considered relevant for the classification and labelling of the substance.

The source substance, 2-Ethylhexyl 4-methoxycinnamate, is also an organic mono-constituent substance used as a sunscreen agent in cosmetics. Details of the source substance purity are not publicly available. However, as a mono-constituent substance a purity ≥80% is required for OMC. Both esters of p-methoxycinnamic acid, IMC and OMC are close structural analogues, differing only in the length of the carbon chain originating from the alcohol. Consequently, the impurity profile is expected to be similar.

3. ANALOGUE APPROACH JUSTIFICATION

As IMC and OMC are structurally similar, ecotoxicity results for the two substances are also expected to be similar. Where data are available on both substances, these ‘anchor points’ are compared in order to assess the similarity of the two substances. Reliable (Klimisch 1 and 2) studies are available for acute toxicity to Daphnia and toxicity to algae for both substances.

A study is available for IMC for acute toxicity to Daphnia following EU method C2, and a filtered emulsion was tested (Bayer 2000). The EC50 value was determined as the geometric mean of the EC0 and EC100 values, 0.28 mg L-1, based on geometric mean measured concentrations. A second study is available for IMC, but as only a results page is available for the study it is assigned a Klimisch score of 4 (Bayer 1990). The test solutions were prepared by weighing the test item into water and treating in an ultrasonic bath, but the test item was not fully dissolved. The EC50 was reported to be 5.6 mg L-1 (geometric mean of EC0 and EC100). OMC was tested in a study conducted according to OECD 202 and due to the low solubility of the test item the Daphnia were exposed to an eluate of the test item (Proprietary 2003). No inhibitory effects were reported at the limit of solubility and the EC50 was reported as >0.027 mg L-1, based on the median analytical recovery rate after 0 and 48 hours. Both IMC and OMC are poorly soluble and this affected the preparation of the test solutions and the conduct of the studies. OMC showed no inhibitory effects at the limit of solubility, whereas effects were observed for IMC, likely due to the slightly higher solubility of the test item.

Toxicity to algae was assessed for IMC in a GLP-compliant study conducted according to EU method C3 (Bayer 2000). The stock solution was prepared by addition of the test item, stirring and then filtering the emulsion to remove undissolved test item. The 72-hour EC50 was 0.2 mg L-1 (growth rate), and the NOEC was 0.06 mg L-1 (cell density), both based on arithmetic mean measured concentrations. A second study is available, but is assigned a Klimisch score of 4 due to very limited method details being available (Bayer 1994). The study followed OECD guideline 201 and reported 72-hour EC50 values of 0.34 mg L-1 and 0.5 mg L-1 (effect not stated). EC10 values of 0.18 mg L-1 and 0.25 mg L-1 (effect not stated) and a NOEC of 0.13 mg L-1 (cell number) were also reported. For OMC, a study is available following OECD guideline 201 (Proprietary 2000). Test solutions were analysed at 0, 24, 48 and 72 hours but most of the measured concentrations were less than the Limit of Detection. An EC50 value of >100 mg L-1, an EC10 of 65 mg L-1 and a NOEC of 32 mg L-1 (all based on nominal concentrations) were reported. A supporting study also following OECD guideline 201, but with no analysis of test concentrations reported nominal EC10 and EC50 values of >100 mg L-1 (Proprietary 2001). There are apparent differences between the results for IMC and OMC, however as the OMC results are based on nominal concentrations and the study where analysis was conducted showed actual test concentrations less than the Limit of Detection, it is difficult to directly compare the results from the studies.

Acute toxicity to fish data are available for both substances, however the data for IMC is assigned a Klimisch score of 4 due to a summary page and not a full test report being available (Bayer 1998). Based on the available results fish are clearly the least sensitive species for both substances. The study with IMC reports a 96-hour LC50 of 2691 mg L-1 (LC0/LC100), and the studies with OMC report LC50 values of 1216 mg L-1 (statistically evaluated from the LC20 and LC100 value) and >100 mg L-1 (BASF 1998, Proprietary 2000). Both substances were tested at concentrations well above their limit of solubility. Results for other ecotoxicity endpoints show that IMC may be more toxic in the aquatic environment than OMC, which is likely to be due to the slightly higher solubility of the test item. For this reason, read across is not used for the most sensitive ecotoxicity endpoints (Daphnia and algae), meaning that classification of IMC is driven by data for the substance itself, and the PNECs are also based on IMC data. However, as fish are the least sensitive endpoint and the available data for IMC itself shows an EC50 value well above those for Daphnia and algae, read across is considered to be appropriate for this endpoint in order to reduce vertebrate testing. As both substances are structurally very similar, toxicity to aquatic organisms is likely to be similar, aside from the differences in solubility.

4. DATA MATRIX
See attached read across justification report attached in the Robust Study Summary.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 1
Test material information:
Composition 1
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
271.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
other: LC20
Effect conc.:
604.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 216.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1 422.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Conclusions:
In the acute toxicity test to Brachydario rerio with the tested substance a LC50 of 1,216.1 mg/L and an LC100 of 1,422.3 mg/L (96h) was
determined. The test was performed according to OECD guideline no. 203.
Executive summary:

The short-term toxicity of the test substance to Brachydanio rerio was tested according to OECD guideline no. 203. The NOEC was 271.3 mg/L, whereas the minimal concentration leading to 100% mortality in the tested species was determined at 1,422.3 mg/L (=LC100). The LC50 (1,261.1 mg/L) was mathematically derived from the LC20 value (604.5 mg/L) and the LC100 value (1,422.3 mg/L).

The target substance and the tested substance are both esters of p-methoxycinnamic acid. Due to this close structural relationship, these two compounds are considered to show comparable ecotoxicological properties. Therefore, the present results are used in a read-across approach to support the findings made in the ready biodegradation study conducted with isopentyl p-methoxycinnamate.

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose:
reference to same study
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
not specified
Principles of method if other than guideline:
no data
GLP compliance:
not specified
Test material information:
Composition 1
Analytical monitoring:
not specified
Details on test solutions:
no details given
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
no details given
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
no details given
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
1 600 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
4 526 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Remarks on result:
other: substance is deposited in droplets at the base of the bowl
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2 691 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
not specified
Basis for effect:
not specified
Remarks on result:
other: LC50= LC0/LC100
Details on results:
no details given
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data

not applicable

Validity criteria fulfilled:
not specified
Conclusions:
In the acute toxicity test to Brachydario rerio with the test substance a LC0 of 1600 mg/L and an LC100 of 4526 mg/L was
determined. As only a result page is available no detailed information on the perfomance of the test (e.g. use of
controls, experminental conditions) or detailed results (e.g. graph of the concentration-mortality curve; statistical data) are given.
Executive summary:

The short-term toxicity of the test item to Brachydanio rerio was tested in a 96h-Test. The highest concentration which did not cause mortality was 1,600 mg /L (=LC0), whereas the minimal concentration leading to 100% mortality in the tested fish was determined at 4,526 mg/L (=LC100).

Description of key information

The test substance is not acutely toxic to fish (LC50 = 1216 mg/L). No effects within water solubility were observed (LC50 > water solubility).

Key value for chemical safety assessment

LC50 for freshwater fish:
1 216 mg/L

Additional information

In the study with isopentyl p-methoxycinnamate itself, the highest concentration which did not cause mortality was determined to be 1,600 mg/L (=LC0), whereas the minimal concentration leading to 100% mortality in the tested fish was determined at 4,526 mg/L (=LC100). The geometric mean of these two values, which is considered to represent the LC50 value, was calculated to be 2,691 mg/L. All tested concentrations are far above the water solubility (WS = 0.8 mg/L). Thus, no effects within the water solubility were observed. As only a result page is available and thus no details on the study are available, the reliability of this study cannot be assessed and therefore a reliability level of 4 (not assignable) is given to this study. Together with the test of a read-across substance, the test is used in a weight of evidence approach.

The short-term toxicity study with the structurally similar 2-ethylhexyl 4-methoxycinnamate with Brachydanio rerio showed the following results: The NOEC was 271.3 mg/L, whereas the minimal concentration leading to 100% mortality in the tested fish was determined at 1,422.3 mg/L (=LC100). The LC50 value (1,261.1 mg/L) was mathematically derived from the LC20 value (604.5 mg/L) and the LC100 value (1,422.3 mg/L). As well for this study, the tested concentrations are far above the water solubility of the substance (WS < 1 mg/L) and no effects within the water solubility were observed.

The LC50 value (1,261.1 mg/L) of 2-ethylhexyl 4-methoxycinnamate is in the same range as the LC50 value (2,691 mg/L) of isopentyl p-methoxycinnamate. Both substances are esters of p-methoxycinnamic acid and therefore show close chemical relationship. The assumption that 2-ethylhexyl 4-methoxycinnamate has comparable ecotoxicological properties for fish is supported by the results of the two short-term toxicity studies with fish. Therefore, both tests were used as Weight of Evidence approach. As worst case the lower LC50 of the two studies is used for chemical safety assessment (LC50 = 1261 mg/L for 2-ethylhexyl 4-methoxycinnamate).

Furthermore the acute tests with daphnia and algae of isopentyl p-methoxycinnamate showed that the tests which were rated as RL4 due to the missing information on the test performance (only result page available) are in a similar range with the results of the available GLP guideline studies. Thus, it is assumed that a GLP guideline study with fish will be in the same range of effects (LC50 > water solubility). Due to animal welfare and as Daphnia and algae showed to be the much more sensitive species, an additional acute fish test is considered as not needed to assess this endpoint. See attached read across justification report fot full details of the read across approach.

Data on marine fish are not available.