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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-26 to 2018-12-11
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:
Adopted 13 April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
solubility in water: < 0.51 mg/L (20°C, pH 7.2) according to study R 014/2017; DMT-dT Phosphoramidite: Water solubility in distilled water (column elution method); WeylChem InnoTec GmbH (see also chapter 4.8)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Limit Test, 100 mg/L (nominal); The concentrations of the test item 5’-O-[bis(4-
methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] were analysed in the duplicate test media samples from the only test concentration and in the duplicate control samples from all sampling times (0, 24 and 48 hours).
- Sampling method: The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director's responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the aged test media of the only test concentration and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment. All samples were diluted by a factor of 2 with acetonitrile. Additional samples of the control and the dilution solvent were taken at each sampling date without any sample treatment.
- Sample storage conditions before analysis: All samples were stored in a freezer (s - 20 °C), protected from light, until analysis was performed. Afterwards the samples were again stored deep frozen (s -20 °C) and will be kept stored up to the date of the final report.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water solubility of test item was indicated to be low (< 0.51 mg/L) and when preparing the fresh media particles were floating on the surface or were lying on the bottom of the flask. To avoid physical effects of undissolved test item on the daphnids, no concentration above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 103.3 mg of test item in 1033 mL test water and 103.1 mg of test item in 1031 mL test water. The stock solution was shaken overhead for 48 hours in the dark to dissolve as much test item as possible. Then, the undissolved test item was separated by filtration (0.45 um cellulose acetate filter) and the filtrate was used as test medium. It is important to remove non-dissolved test substance from the test medium to avoid physical effects of undissolved test item on the daphnids. According to OECD 23, filtration is a recommended procedure.
The test media was prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
- Eluate: N/A
- Differential loading: N/A
- Controls: In the control, test water was used without addition of the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A (test item disolved in medium)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): During preparation: see above; Appearance of the Test Item in Test Medium: No remarkable observations.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Justification for species other than prescribed by test guideline: N/A
- Source: The daphnids introduced in the test were taken from ibacon's in house laboratory culture
- Age at Test Start: From 2.75 to 19.25 hours old
- Feeding during test: No
- Reference Item: For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
ACCLIMATION
Acclimatisation was not necessary, since the test was performed in the same medium as the culturing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
2.5 mmol/L (= 250 mg/L) as CaCO3
Test temperature:
20.2 to 20.7 °C in the freshly prepared media;
20.6 to 21.0°C in the aged test media
pH:
7.8 to 8.3 in the freshly prepared media;
7.8 to 8.0 in the aged test media
Dissolved oxygen:
6.1 to 8.9 mg/L in the freshly prepared media;
8.3 to 8.7 mg/L in the aged test media
Nominal and measured concentrations:
A filtrate of a supersaturated test solution, i.e. 100 mg test item/L (nominal) and a control.
The water solubility of test item was indicated to be low (< 0.51 mg/L) and when preparing the fresh media particles were floating on the surface or were lying on the bottom of the flask. The measured concentration were below the detection limit of 0.01 mg/L although the substance was stirred for 48 h in the medium.
Details on test conditions:
TEST SYSTEM
- Test vessel: Type and Size: Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Type (delete if not applicable): open
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static, no flow-through
- Renewal rate of test solution (frequency/flow rate): every 24 h
- Introduction of Daphnids: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in 60 mL test medium
- Replicates: The test was performed with four replicates per treatment group.
- Exposure Time: 48 hours
- Test Procedure: A semi-static test system with a test medium renewal on Day 1 was chosen to keep the concentrations of test item as constant as possible in the test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates): 4
Test Units
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Test medium: Elendt M4
- Culture medium different from test medium: No
- Light Regime: 16 h light : 8 h dark
- Light Intensity: The light intensity was 350 to 710 lux (measured once during the test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility. The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae). The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 100, 10, 1 and 0.1 mg/L
- Results used to determine the conditions for the definitive study: Yes

Pre-Experiments:Pre-experiments were performed to determine a suitable concentration range for the main test and to establish suitable methods for the preparation and analytical measurement of the test solutions. The test item was stirred 48 hours and the undissolved test item was separated by filtration (0.45 µm cellulose acetate filter). 10 daphnia were exposed to the filtrate of 100 mg/L and dilutions thereof of 1:10, 1:100 and 1:1000. The test media were renewed after 24 hours. No effect on the daphnia was observed.
The pre-experiments were not performed in compliance with the GLP-Regulations and were excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Filtrate of nominal 100 mg test item/L
Basis for effect:
mobility
Remarks on result:
not determinable because of methodological limitations
Remarks:
Concentration was below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Filtrate of nominal 100 mg test item/L
Basis for effect:
mobility
Remarks on result:
not determinable because of methodological limitations
Remarks:
Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Filtrate of nominal 100 mg test item/L
Basis for effect:
mobility
Remarks on result:
not determinable because of methodological limitations
Remarks:
Concentration must be below LOD of 0.01 mg/L though a supersatured solution of the test item was prepared.
Details on results:
- Behavioural abnormalities: No
- Observations on body length and weight: No
- Other biological observations: No
- Mortality of control: No mortality occurred in the control
- Other adverse effects control: No
- Abnormal responses: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: See details on test solution. During the test no irregularities in the medium were observed.
- Effect concentrations exceeding solubility of substance in test medium:
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate performed in January 2018 (study code 88299220), the EC50 after 24 hours was determined to be 1.06 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
In the reference test performed in June 2017 (study code 88298220) with the reference item potassium dichromate, the EC50 after 24 hours was determined to be 1.58 mg test item/L and the EC50 after 48 hours was determined to be 1.07 mg test item/L.
Reported statistics and error estimates:
No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Validity Criteria of the Study

 

Control Immobilisation Rate:

0 %, no daphnid showed signs of disease or stress; thus the validity criterion was met.

 

Dissolved Oxygen Concentration:

³ 8.3 mg O2/L in the control and test vessels at the end of the test; thus validity criterion was met.

 

 

 

 

Biological Results

 

 

Signs of Intoxication after 48 Hours:


After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of the filtrate of 100 mg test item/L (see Table 1).

 

 

 

 

 

Table 1. Influence of 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] on the Mobility of Daphnia magna

 

 

Treatment group

% of immobilised daphnids after

 

[mg test item/L]

24 hours

48 hours

 

Control

0

0

 

Filtrateof 100

0

0

 

The EC50, EC20and EC10could not be calculated due to the low effects of the test item. Therefore the 72-hourEC50, EC20and EC10was determined to be higher than the limit of water solubility of the test item, represented by the filtrate of nominal 100 mg test item/L.

TheNOECwas determined to be at least the filtrate of 100 mg test item/L. The LOEC wasestimated to behigherthan the filtrate of 100 mg test item/L.

 

 

 

 

Analytical Results

 

 

 

Table 2. Summary of Analytical Results

 

Sample description

 

Nominal

% of

RSD

[mg test item/L]

nominal1

[%]

n

 

 

 

 

Control

n.a.

n.a.

8

Filtrate of 100

<LOD

n.a.

8

 

 

 

 

 

1mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

LOD (=Limit of Detection): 0.01 mg test item/L

 

 

Validity criteria fulfilled:
yes
Conclusions:
In an acute toxicity test conducted as Limit test according to the OECD guideline 202 Daphnia magna were exposed to 100 mg/L (nominal) 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite]. As the test substance is hardly soluble, less than the limit of detection was dissolved in the medium (LOD = 0.01 mg/L), although the test solution was stirred for 48 hours. No effect was observed.
Executive summary:

The 48-hr-acute toxicity of 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] to Daphnia magna was studied under semi-static conditions. Test species were exposed to control and test chemical at nominal concentration of 100 mg/L. for 48 hr. As the test substance is hardly soluble, less than the limit of detection was dissolved in the medium (LOD = 0.01 mg/L), although the test solution was stirred for 48 hours. Mortality/immobilisation and sublethal effects were checked daily.  No effect were observed up to the limit of the test item solubility.

 

Based on the results of this study, 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] would be classified as not toxic to D. magna in accordance with the classification system of the EU.

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates. 

 

Results Synopsis

 

Test Organism Age (e.g. 1stinstar): < 24 h

Test Type (Flowthrough, Static, Static Renewal): Static

Table1. Summary of Biological Results

Nominal Concentration

% of immobilised daphnids after

[mg test item/L]

24 hours

48 hours

Control

0

0

Filtrateof 100

0

0

EC50[mg test item/L]:

> filtrate 100

> filtrate 100

95 % CI [mg test item/L]:

n.d.

n.d.

EC20[mg test item/L]:

> filtrate 100

> filtrate 100

95 % CI [mg test item/L]:

n.d.

n.d.

EC10[mg test item/L]:

> filtrate 100

> filtrate 100

95 % CI [mg test item/L]:

n.d.

n.d.

NOEC [mg test item/L]:

≥filtrate 100

≥filtrate 100

LOEC [mg test item/L]:

> filtrate 100

> filtrate 100

 

Endpoint(s) Effected:mobility, mortality

Description of key information

The toxicity of 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] was assessed in a 48-hr acute toxicity test (limit test design) with Daphnia magna according to OECD test guideline 202 and under GLP. No effects were observed up to the limit of water solubility.

Key value for chemical safety assessment

Additional information