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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013-10-10 - 2013-06-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP OECD TG 211 study without deviations on suitable read-across substance
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The structural analogue 1,2,3-Propanetriol, glycidyl ethers is a mixture of different components with average 2-3 epoxy functional groups per molecule, to be more accurate, with 2,3-epoxypropyl side chains. Those are identical to the side chain of 2,3-epoxypropyl isopropyl ether, which is considered to be the toxicologically more relevant functional group. Also, the isopropyl chain is related to the partially substituted propane moiety of 1,2,3-Propanetriol, glycidyl ethers. Both substances are rather small molecules, indicating a similar distribution pattern, which is supported by the liquid state, high water solubility, and low logPow, especially in ecotoxicity tests. They are both not readily biodegradable and sufficiently hydrolytically stable over the usual short-term exposure period in ecotoxicity testing. In consequence, 1,2,3-Propanetriol, glycidyl ethers may serve as a read-across substance for 2,3-epoxypropyl isopropyl ether.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source Chemical: 1,2,3-Propanetriol, glycidyl ethers, CAS 90529-77-4
Target Chemical: 2-(isopropoxymethyl)oxirane, CAS 4016-14-2

3. ANALOGUE APPROACH JUSTIFICATION
As stated above, the analogue (source) consists of components with average 2-3 2,3-epoxypropyl side chains. Those are identical to the side chain of 2,3-epoxypropyl isopropyl ether, which is considered to be the toxicologically more relevant functional group. Both substances are rather small molecules, indicating a similar distribution pattern, and especially relevant for ecotoxicity tests, they both have a high water solubility and low logPow. There is in general no indication that both substances may not pass biological membranes except a possible direct protein binding via nucleophilic addition via the epoxy group, which is however a common mechanism. Both substances are sufficiently stable during the average duration of short-term ecotoxicity tests or when performed in a flow-through design: They are both not readily biodegradable and sufficiently hydrolytically stable.
With regard to the comparability of the ecotoxicity data, ECOSAR Version 1.11 by US EPA predictions were used for comparison as these values are easily available for both substances. For IPGE, there are Class-specific Estimations available, both enormous differing in the magnitude of the values. However, in general, the values obtained for the source chemical are in between the values derived for IPGE (Epoxides, mono, or Neutral Organics), showing that there is no definitive reason to believe that the values derived from actual IPGE testing would largely differ frim the current ones for CAS 90529-77-4.
In consequence, 1,2,3-Propanetriol, glycidyl ethers may serve as a read-across substance for 2,3-epoxypropyl isopropyl ether.

4. DATA MATRIX
See Attachment
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
read-across source
Remarks:
source record
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Federal Public of Health, Bern
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
PHYSICO-CHEMICAL PROPERTIES
- Melting point: < -20°C
- Boiling point: substance decomposes before boiling occurs: decomposition temperature ≥ 181°C (at 101.325 kPa)
- Vapour pressure: 0.0392 Pa (estimated)
- Water solubility (under test conditions): 20 -30 g/L
- Henry's law constant: 2.15E-9 Pa*m³/mol (Bond Method), 2.37E-14 Pa*m³/mol (Group Method) (estimated)
- log Pow: -1.94 (estimated)

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: not readily biodegradable
- Other: hydrolytically stable (estimated)
Analytical monitoring:
yes
Details on sampling:
- Water quality criteria: At the beginning and end of each test medium renewal period, the pH and dissolved oxygen concentrations were measured in one replicate of each test concentration and of the control. At the same time, the water temperature was measured in one of the control replicates. Additionally, the room temperature was continuously monitored. The test media appearance was visually inspected and recorded at the beginning and end of each test medium renewal period.
- Analysis of test substance concentration: Duplicate samples from the freshly prepared test media of all concentrations and from the control at one treatment period of the first, second and last week of the test (Day 0, 7 and 16, respectively)
- Maintenance of the test substance concentration: At the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).
- Stability samples (in duplicate): a) from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal period. b) samples incubated during the renewal period under test conditions, without food and daphnids
- Sample storage conditions before analysis: Methanol (v/v; 9/1) and 10 µL formic acid were added to each sample taken in order to stabilize them during the storage period. Till date of analysis, samples were deep frozen at -20 °C.
Vehicle:
no
Details on test solutions:
Prior to the start of the experiment and prior to each test medium renewal, the test medium of the highest nominal concentration (100 m/L) was freshly prepared by completely dissolving 120 mg of the test substance (dosed in the range of 119.8 - 121.1 mg) in 1200 mL of the test water or 140 mg of the test substance (dosed in the range of 139.9 - 140.3 mg) in 1400 mL of test water using stirring for 15 min at room temperature. The test medium of the highest test concentration was used on a series of dilution with test water to prepare the lower concentrated test media.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Waterflea
- Strain/clone: STRAUS
- Source: University of Sheffield, UK in 1992 ("Clone 5") - since this date, the clone is successfully bred at Harlan Laboratories.
- Feeding during test
- Food type: Food mixture containing a suspension of Scenedesmus subspicatus (freshly grown) and a fish food suspension. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, Melle, Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. 400 mL of the supernatant was diluted 1:1 with test water and boiled. This suspension was fed in addition to the algal food, because a toxic effect of the test substance on the algae could not be excluded. Furthermore, thereby the amount of algal food could be reduced in order to prevent an increase of the pH values in the test media due to algal growth.
- Amount: 0.20 mg TOC/Daphnia/day (Determined using a Shimadzu TOC 5000A Analyser)
- Frequency: each working day (Monday through Friday)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
No post exposure observation period described.
Hardness:
2.5 mmol/L (250 mg/L as CaCO3)
Test temperature:
20 - 21 °C
pH:
Control: 7.7 - 8.1
1.0 mg/L: 7.7. - 8.1
3.2 mg/L: 7.7 - 8.2
10 mg/L: 7.8 - 8.2
32 mg/L: 7.7 - 8.2
100 mg/L: 7.7 - 8.2
Dissolved oxygen:
Control: 8.0 - 9.6 mg O2/L
1.0 mg/L: 8.0 - 9.6 mg O2/L
3.2 mg/L: 8.0 - 9.6 mg O2/L
10 mg/L: 8.1 - 9.6 mg O2/L
32 mg/L: 8.0 - 9.6 mg O2/L
100 mg/L: 8.1 - 9.5 mg O2/L
Salinity:
Not applicable.
Nominal and measured concentrations:
0, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers containing 80 mL of test medium
- Type: closed
- Aeration: none
- Renewal rate of test solution: every 48 or 72 hours (Days 2, 5, 7, 9, 12, 14, 16 and 19 - every Monday, Wednesday and Friday)
- No. of organisms per vessel: 1 (10 daphnids/treatment)
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Main compounds: 294 mg/L CaCl2 x 2 H2O, 123 mg/L MgSO4 x 7 H2O, 5.8 mg/L KCl, 65 mg/L NaHCO3, 10 mg/L Na2SiO3 x 9 H2O, 0.27 mg/L NaNO3, 0.14 mg/L KH2PO4, 0.18 mg/L K2HPO4
- Trace elements: 125 µg/L H3BO3, 50 µg/L FeSO4 x 7 H2O, 25 µg/L MnCl2 x 4 H2O, 12.5 µg/L LiCl, 12.5 µg/L RbCl, 12.5 µg/L SrCl2 x 6 H2O, 3.1 µg/L NaBr, 6.3 µg/L Na2MoO4 x 2 H2O, 1.6 µg/L CuCl2 x 2 H2O, 6.3 µg/L ZnCl2, 2.5 µg/L CoCl2 x 6 H2O, 2.5 µg/L KI, 1.0 µg/L Na2SeO3, 0.3 µg/L NH4VO3, 625 µg/L Na2EDTA x 2 H2O
- Vitamins: 75 µg/L Thiamine HCL, 1.0 µg/L Cyanocobalamine (B12), 0.75 µg/L Biotin (B6)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 light-dark-cycle with a 30 min transition period
- Light intensity: approx. 390 - 560 Lux

RANGE-FINDING STUDY
The selection of the test concentrations was based on a range-finding test and on results of a pre-experiment to determine the solubility of the test substance.
Reference substance (positive control):
not required
Remarks:
according to the applied guidelines
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
8.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
8.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Test media appearance: No remarkable observations were made. All test media were clear solutions throughout the test medium renewal periods.
- Analytical results: The measured test substance concentrations in the freshly prepared test media of the nominal concentrations of 10 and 32 mg/L were between 77 and 97 % of the nominal values at the start of the test medium renewal periods.
In the aged test media samples without food, the measured concentrations were between 68 and 82 % of the nominal values. In the aged media samples with food, the measured concentrations were between 69 and 81 % of the nominal values. Similar recoveries were determined in the aged test media samples incubated without food and daphnids.
- Stability of the test substance: The test substance was not stable during the test medium renewal periods of two and three days. Therefore, all reported biological results are related to the mean measured concentrations. These were calculated as the time-weighted means over the measurements at the start and end of the renewal periods. For the end of the renewal periods, the samples with food were taken into account.
- Survival: In the control and up to and including the highest test concentration, the survival of the test animals at the end of the experiment was at least 90 % or higher. Mortality up to 20 % is regarded as natural and tolerated by the test guideline. Thus, the survival of Daphnia magna over 21 days was not affected by the test item up to and including the highest nominal test concentration of 100 mg/L.
_ Offspring: The first young offspring released from their parent animals were recorded at the test concentrations of 1.0 - 32 mg/L at observation on Day 9. At the highest test concentration a delay of the first brood was observed (first offspring observed on Day 12 of exposure). Thus, the time of the first brood was not affected by the test substance up to and including the mean measured concentration of 26 mg/L.
- Reproduction rate: The mean reproduction rate of the daphnids in the control was 93 +/- 20 living offspring per surviving adult (mean +/- standard deviation). No statistically significant inhibitory effect of the test substance on the mean reproduction rate was determined up to and including the mean measured concentration of 26 mg/L and the highest test concentration, the mean reproduction rates of surviving daphnids was statistically significantly reduced to an average of 69 and 50 living offspring (75 and 54 % compared to the control), respectively. The EC50 was clearly higher than the highest tested concentration of nominal 100 mg/L, because none of the responses exceeded 50 %.
- Conclusion on observations: With the exception of the reduced reproduction rates, no visible abnormalities were observed at the test animals during the experiment.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
The mean reproduction rates of the daphnids at the test concentrations were compared to the control by multiple Williams t-tests.
Reference:
Williams, D.A. (1971): A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103-117
Williams, D.A. (1972): The comparison of several dose levels with a zero dose control. Biometrics, 28, 519 - 531
Validity criteria fulfilled:
yes
Remarks:
Validity criteria of the applied test guideline were fulfilled.
Conclusions:
The study report describes a valid guideline study conducted under GLP compliance on 1,2,3-Propanetriol, glycidyl ethers. Based on effects on survival and reproduction, the highest concentration tested without toxic effects (NOEC) after 21 days exposure duration was the mean measured concentration of 8.3 mg/L. Hence, the results do not trigger classification as hazardous to the aquatic environment according to Regulation 1272/2008. The test item was considered suitable for read-across to 2,3-epoxypropyl isopropyl ether, hence, it can be concluded that 2,3-epoxypropyl isopropyl ether can be regarded as non-toxic to daphnids, too.
Executive summary:

The toxic effects of 1,2,3-Propanetriol, glycidyl ethers towards reproduction and survival of the freshwater invertebrate Daphnia magna was investigated according to OECD Guideline 211 / EU Method C.20. The experiment was conducted as semi-static test with renewals every 48 or 72 h. The total exposure duration was 21 days. The water temperature was maintained at 20 - 21 °C and the dark-light-cycle was set at 8 h/16 h with 30 min transition periods. Feeding of the test animals was performed daily each working day with a food mixture containing a suspension of the green algae species Scenedesmus subspicatus and a fish food suspension. The following nominal concentrations of the test substance were tested: 1.0, 3.2, 10, 32 and 100 mg/L. 10 replicates per concentration were prepared. At the start, 10 daphnids per treatment were employed, whereby each animal was kept individually in a glass beaker. Additionally, a control was tested in parallel (test water only). The selection of the test concentrations was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test substance. On Days 0 - 2 and thereafter three times per week, the test replicates were observed for immobility and for the number of living and dead offspring as well as for the presence of aborted eggs. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the experiment. The mean reproduction rates at the test concentrations were compared to the control by Williams t-tests. An EC50 value, based on reproduction rate, could not be calculated since none of the responses exceeded 50 %. The value was therefore determined directly from the raw data. High Performance Liquid Chromatography with Mass Spectrometry (HPLC-MS/MS) was the analytical method of choice. The measured test substance concentrations in the freshly prepared test media of the nominal concentrations of 10 and 32 mg/L were between 77 and 97 % of the nominal values at the start of the test medium renewal periods. In the aged test media samples without food, the measured concentrations were between 68 and 82 % of the nominal values. In the aged media samples with food, the measured concentrations were between 69 and 81 % of the nominal values. Similar recoveries were determined in the aged test media samples incubated without food and daphnids. Consequently, the presence of test organisms and organic food had no impact on the test substance dissolved in test water. No remarkable observations on the test media were recorded. All test media were clear solutions throughout the test medium renewal periods. The test substance was not stable during the test medium renewal periods of two and three days. Therefore, all reported biological results are related to the mean measured concentrations. These were calculated as the time-weighted means over the measurements at the start and end of the renewal periods. For the end of the renewal periods, the samples with food were taken into account. In the control and up to and including the highest test concentration, the survival of the test animals at the end of the experiment was at least 90 % or higher. Mortality up to 20 % is regarded as natural and tolerated by the test guideline. Thus, the survival of Daphnia magna over 21 days was not affected by the test item up to and including the highest nominal test concentration of 100 mg/L. The first young offspring released from their parent animals were recorded at the test concentrations of 1.0 - 32 mg/L at observation on Day 9. At the highest test concentration a delay of the first brood was observed (first offspring observed on Day 12 of exposure). Thus, the time of the first brood was not affected by the test substance up to and including the mean measured concentration of 26 mg/L. The mean reproduction rate of the daphnids in the control was 93 +/- 20 living offspring per surviving adult (mean +/- standard deviation). No statistically significant inhibitory effect of the test substance on the mean reproduction rate was determined up to and including the mean measured concentration of 26 mg/L and the highest test concentration, the mean reproduction rates of surviving daphnids was statistically significantly reduced to an average of 69 and 50 living offspring (75 and 54 % compared to the control), respectively. The EC50 was clearly higher than the highest tested concentration of nominal 100 mg/L, because none of the responses exceeded 50 %. As conclusion on the mentioned observations, it can be stated, with the exception of the reduced reproduction rates, no visible abnormalities were observed at the test animals during the experiment. The final results, based on survival and reproduction of the test animals can be reported as followed: LOEC(21d): 8.3 mg/L and NOEC(21d): 26 mg/L as mean measured concentrations. The EC50 value was clearly higher than the highest tested concentration of nominal 100 mg/L, because none of the responses exceeded 50 %.

Description of key information

Long-term toxicity to aquatic invertebrates: 21d NOEC = 8.3 mg/L, LOEC = 26 mg/L, EC50 > 100 mg/L, based on reproduction and mortality for daphnia magna (OECD 211, GLP, semi-static, on 1,2,3-Propanetriol, glycidyl ethers)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
8.3 mg/L

Additional information

There is a valid, well-documented GLP OECD 211 guideline study without relevant deviations on the structural analogue 1,2,3-Propanetriol, glycidyl ethers available, assessed with Klimisch 2. In this study, the following values were determined: 21d NOEC = 8.3 mg/l, LOEC = 26 mg/l, EC50 > 100 mg/l. The results can be considered as sufficiently reliable to assess the toxicity of 2,3-epoxypropyl isopropyl ether to daphnids. The current results do not trigger classification of ,2,3-Propanetriol, glycidyl ethers and hence 2,3-epoxypropyl isopropyl ether as hazardous to the aquatic environment.

The database is of high quality, and the tonnage-driven data requirements under REACH are fully met, as, according to REACH Annex VII column 1 and 2, testing for short-term toxicity may be replaced by long term testing, hence, no data gap was identified.