N-benzoyl-5'-O-(p,p'-dimethoxytrityl)-2'-deoxycytidine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-22 to 2018-03-27

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:

13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling: The samples were filled into 10 mL glass vials. To each vial, 5 mL acetonitrile (ACN) was added and samples were stored in the freezer (≤ -18 °C). The sampling was conducted according to the following specification:
-After 0 h, 24 h and 48 h exposure, all replicates of NC, A, B, C, D and E were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
For each sampled treatment, one of the samples from 0 h, 24 h and 48 h was sent to the analytical laboratory at the test site menal GmbH for chemical analysis. The remaining samples were stored as retain samples in the freezer (≤ -18 °C) until finalisation of the study at Hydrotox GmbH.
- Storage: At the test site menal GmbH all samples were stored in a freezer at - 20 °C ± 10 °C until analysis.
- Shipment: Personal handover between Hydrotox GmbH and menal GmbH

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution was prepared as Water Soluble Fraction (WSF) by adding 400.1 mg test item to 1000 mL test medium and shaking for 48 h using an overhead shaker at 19.9 – 22.5 °C in the dark. The WSF was filtered through a fibre-glass filter with a retaining range till 0.6 μm (MN 85/70 BF, Marchery-Nagel, Düren, Germany). The filter was prepared by rinsing with purified water and preconditioning with ca. 100 mL WSF (which was discarded) to reduce adsorption of the test item. This filtered stock solution was used as highest test item loading rate in the test.Four replicates, with five daphnids each, were prepared for each test item treatment and the negative control. The further test loading rates were prepared by diluting the stock solution
- Control: The negative control (NC; test medium) was treated in the same way as the test item solution.
- No vehicle

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: The test organism Daphnia magna Straus originates from the clone breeding of the Federal Environment Agency, department IV 2.4, Berlin, and has been cultivated at Hydrotox GmbH since October 2012
- Feeding during test: no

CULTURING
The daphnids are cultured at 20 ± 2 °C with 16 h light : 8 h dark and placed into fresh Elendt M4 medium twice a week. They are kept at approx. 10 animals / 200 mL and fed on workdays with Desmodesmus subspicatus algae corresponding to 0.1 mg C × daphnid-1 × day-1. The test organisms used were 1.0 - 22.5 h old at the start of the test.
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany; Lot no.: MKBF2111V, expiration date: January 2018). The recent quality testing was performed in August 2017 with EC50 (24h) = 1.77 mg/L (CL 95 %: 1.63 – 2.05 mg/L), which is in the declared range of 0.6 – 2.1 mg/L (24 h) according to OECD 202.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

endpoints observed after 24 and 48 hours

Hardness:

not reported

Test temperature:

19.4 - 20.9 °C

pH:

7.6 - 7.9 (control); 7.7 - 8.3 (test item)

Dissolved oxygen:

7.8 - 8.7 mg/L (control); 7.8 - 8.6 mg/L (test item)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

25, 50, 100, 200, 400 mg test item/L (nominal) and control (0 mg test item/L)
0.112, 0.225, 0.331, 0.561, 1.225 mg test item/L (meas., geom. mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers 50 mL
- Type of flow-through (e.g. peristaltic or proportional diluter): static, no flow-through
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4"), according to OECD 202
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Lowest and no observed effect concentrations of test item on immobility and mortality

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. 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.
- Test concentrations: A preliminary test without GLP was performed before the start of this GLP-study. Nominal loading rates of 50, 100 and 200 mg/L test item were tested and resulted in 0, 20 and 30 % immobility (48 h), respectively.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.612 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.108 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.112 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

0.905 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

> 1.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.225 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities, on body length and weight etc.: not reported; Immobility was dose-dependent and ranged from 5% at 0.112 mg/L to 65% at 0.561 mg/L after 48 hours.
- Mortality of control: 0 %
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the water solubility of the substance is below 0.1 mg/L at pH 7.1 and a temperature of 20 °C. (see section 4.8 in the present dossier).
- Effect concentrations exceeding solubility of substance in test medium: yes, EC50 = 0.612 mg/L, LOEC = 0.225 mg/L, NOEC = 0.112 mg/L

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The effect occurrence after 24 h and 48 h exposure was calculated. With the statistical software ToxRat Professional 3.2.1 (ToxRat Solutions GmbH, Alsdorf, Germany), ECx and LOEC/NOEC were determined. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

- ANALYTICAL RESULTS: In the control, the test item was not detected. The measured test item concentrations are not within ± 20 % of the nominal loading rate. According to OECD 202 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations. Therefore, the geometric mean of all three measurement times (0 h, 24 h and 48 h) was calculated. The test item was shaked in test medium for 48 h. Compared to the water solubility in distilled water (i.e. < 0.1 mg/L at pH 7.1 and 20 °C, see section 4. 8 of this dossier) the solubility of the test item in Elendt M4 medium is slightly higher than in distilled water which can be explained by interactions with the salts contained in the medium.

The chemical analysis was conducted using the method with the lowest LOQ possible with the available equipment at the test site. The signal to noise ratio would be too low for a lower LOQ (in the range of or below the Limit of detection). Table 1 presents the results of the chemical analysis.

Table 1. Analytically measured test item concentrations after 0 h, 24, h, and 48 h of exposure. LOQ = 1 mg/L.

Nominal test item loading rate (mg/L)	Sample	Sampling time (h)	Measured test item concentration (mg/L)	Geometric mean test item concentration (mg/L)
NC	NC1	0	<LOQ	--
NC	NC2	24	<LOQ	--
NC	NC3	48	<LOQ	--
25	A1	0	<LOQ (0.217)	<LOQ (0.112)
25	A2	24	<LOQ (0.093)	<LOQ (0.112)
25	A3	48	<LOQ (0.069)	<LOQ (0.112)
50	B1	0	<LOQ (0.460)	<LOQ (0.225)
50	B2	24	<LOQ (0.159)	<LOQ (0.225)
50	B3	48	<LOQ (0.155)	<LOQ (0.225)
100	C1	0	<LOQ (0.923)	<LOQ (0.331)
100	C2	24	<LOQ (0.219)	<LOQ (0.331)
100	C3	48	<LOQ (0.180)	<LOQ (0.331)
200	D1	0	1.89	<LOQ (0.561)
200	D2	24	<LOQ (0.339)	<LOQ (0.561)
200	D3	48	<LOQ (0.276)	<LOQ (0.561)
400	E1	0	4.35	<LOQ (1.225)
400	E2	24	<LOQ (0.860)	<LOQ (1.225)
400	E3	48	<LOQ (0.491)	<LOQ (1.225)

- BIOLOGICAL RESULTS: All test organisms were immobilised at nominal test loading rate of 400 mg/L (see Table 2).

Table 2. Immobility after 24 h and 48 h exposure

Nominal test item loading rate (mg/L)	Geometric mean test item concentration (mg/L)	Immobility, 24 h (%)	Immobility, 48 h (%)
NC	--	0	0
25	<LOQ (0.112)	0	5
50	<LOQ (0.225)	0	15
100	<LOQ (0.331)	0	45
200	<LOQ (0.561)	30	65
400	<LOQ (1.225)	100	55

- VALIDITY CRITERIA:

-Immobilisation of the test organisms in the control at the end of the test was 0 % and therefore ≤ 10 %.

-Dissolved oxygen concentration in the control and the test item treatments at the end of the test was ≥ 7.8 mg/L and therefore ≥ 3 mg/L.

Therefore, the test is valid according to OECD Test Guideline 202 (13 April 2004).

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test according to the OECD guideline 202 with Daphnia magna an EC50 of 0.612 mg/L (meas., geom. mean) was determined for N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine/L.

Executive summary:

The 48-hr-acute toxicity of N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine to Daphnia magna was studied under static conditions. Daphnids were exposed to control, and test chemical at measured concentrations of 0, 0.112, 0.225, 0.331, 0.561, 1.225 mg/L for 48 hours. Mortality, immobilization and sublethal effects were observed daily. The 48– hour EC50 was 0.612 mg a.i./L. The 48-hr LOEC based on immobilization was 0.225 mg/L. The sublethal effect included was immobility.

The initial concentration and the maintenance of the exposure concentrations during the test were verified in the analytical part. As the measured test item concentrations are not within ± 20 % of the nominal loading rate, according to OECD 202 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.

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. 1st instar): < 24 h

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

 

NOEC: 0.112 mg a.i./L (based on LOQ)

LOEC: 0.225 mg a.i./L

EC50: 0.612 mg a.i./L

 

Endpoint(s) Effected: mobility, mortality

Description of key information

The toxicity of N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine was assessed in a 48-hr-acute toxicity test with Daphnia magna according to OECD test guideline 202 and GLP. The following effect concentrations were determined:

NOEC: 0.112 mg a.i./L

LOEC: 0.225 mg a.i./L

EC₅₀: 0.612 mg a.i./L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.612 mg/L