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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-09-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test not according to GLP and limited details given.
Reason / purpose:
reference to same study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
not specified
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
not specified
GLP compliance:
no
Test material information:
Composition 1
Analytical monitoring:
not specified
Details on sampling:
no data
Details on test solutions:
The test substance was directly weighed at concentrations of 25, 250, and 2,500 mg and applicated to 250 mL of test medium(mixture of water,
culture medium and activated sludge) to receive the final test concentrations of 100, 1000, and 10000 mg/L. 1 Liter of the test medium consists of 32 mL culture medium (prepared according to ISO 8192-1986, capture 4.2), 50 mL activated sludge (6 g/L TS), and 918 mL water.
One blank control (250 mL test medium without adding the test substance) and two different concentrations of a positive control, 1,000 mg/L and 20 mg/L (0.25 mg and 5.0 mg of the reference substance added to 250 mL of the test medium) were additionally tested .
Test organisms (species):
activated sludge
Details on inoculum:
- cultivation of the inoculum: in a 3 L laboratory installation
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no data
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):
yes
Remarks:
3,5-Dichlorphenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
no details given
Results with reference substance (positive control):
The validity of the test was checked by means of graphical analysis of the reference substance. A significant and concentration dependent decrease of the respiration rate or rather an increasing repiration inhibition after 3 h of exposure compared to the control was observed.

Reported statistics and error estimates:
no data

 

test substance

reference substance

control

autoxidation

net weight of the test substance [mg]

25

250

2500

0.25

5.0

0.0

2500

test concentration [mg/L]

100

1,000

10,000

1,0

20,0

0,0

10,000

respiration rate [mg/L*h] 

21

21

18

17

6

20.1

0.0

respiration inhibition [%]

0

0

10

16

70

-

-

Conclusions:
The highest tested concentration of 10,000 mg/L, led to an inhibition of the respiration of the tested inoculum of 10%.
Executive summary:

The toxicity of the test substance to microorganisms was tested in the activated sludge respiration inhibition test (according to OECD guideline no. 209). Three different concentrations as well as a positive control (3,5-dichlorophenol) in 2 different concentrations and a vehicle control (inoculum without adding any substance) were tested. No significant effect on the respiration rate (compared to the control with untreated inoculum) was observed after 3 h of exposure to 100 and 1,000 mg/L. At the highest test concentration of 10,000 mg/L, the respiration rate was decreased by 10% compared to that of the control.

An EC50 could not be determined, but it can be deduced form the experiment that this value must be higher than 10,000 mg/L.

The positive control compound induced an inhibition of respiration of 16% and 70%, when tested at the concentrations of 1 and 20 mg/L, respectively, thereby ensuring the validity of the test system and the sensitivity of the activated sludge used in this test.

 

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
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 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Concentration of inoculum: 1 g/l referred to the dry weight
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Reference substance (positive control):
yes
Remarks:
3,5- Dichlorphenol
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
No inhibition of respiration was observed up to a concentration of 1000  mg/l.
Conclusions:
No inhibition of respiration was observed up to a concentration of 1,000 mg/L.
Executive summary:

The toxicity of the test substance was tested in the activated sludge respiration inhibition test (according to OECD guideline no. 209).

A NOEC of >1,000 mg/L was determined after an exposure period of 30 min.

 

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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.

Reliable activated sludge respiration inhibition studies are available for both IMC and OMC, both following OECD guideline 209 (Bayer 1988, BASF 1997). For IMC, 10% inhibition was reported at the highest test concentration of 10,000 mg L-1, and the 3-hour EC50 was reported as >10,000 mg L-1. For OMC, no inhibition occurred up to the highest test concentration of 1000 mg L-1, and a 30 minute NOEC of >1000 mg L-1 was reported. Both IMC and OMC showed similar results for toxicity to microorganisms, as expected based on their structural similarity, and therefore the results for OMC are used to support the results for IMC.

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
GLP compliance:
yes
Test material information:
Composition 1
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Conclusions:
No inhibition of respiration was observed up to a concentration of 1,000 mg/L.
Executive summary:

The toxicity of the test substance was tested in the activated sludge respiration inhibition test (according to OECD guideline no. 209).

A NOEC of >1,000 mg/L was determined after an exposure period of 30 min.

 

Description of key information

No inhibition of respiration was observed up to a concentration of 1,000  mg/L (NOEC). The EC50 is > 1000 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of isopentyl p-methoxycinnamate to aquatic micro-organisms was tested in an activated sludge respiration inhibition test according to OECD 209. In this test three different concentrations (100, 1,000, and 10,000 mg/L) as well as a positive control (3,5-dichlorophenol at 1 and 20 mg/L) and a control (inoculum without adding any substance) were tested using an exposure period of 3 h. An inhibition of 10% was observed at the highest test concentration of 10000 mg/L. Thus, the NOEC(3h) is 1000 mg/L and the EC50(3h) is > 10000 mg/L.

A supporting study is also available for the read across substance2-ethylhexyl 4-methoxycinnamate. The read across substance has a similar structure to isopentyl p-methoxycinnamate, and measured physico-chemical data for the two substances is also similar. The toxicity of the two substances in the environment is therefore likely to be similar and read across between the two is considered to be acceptable.

The microbial toxicity of the structurally similar substance 2-ethylhexyl 4-methoxycinnamate was assessed in a GLP study according to OECD 209. In the 30 min exposure period no inhibition of respiration was observed up to the highest tested concentration of 1000 mg/L. The NOEC(30min) is >= 1000 mg/L. The result of the activated sludge respiration inhibition test of 2-ethylhexyl 4-methoxycinnamate supports the assessment for isopentyl p-methoxycinnamate.