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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- 4 sampling time points of at each of 2 test concentrations (8 samples). The number of quality controls was six (three at the beginning and the end of an assay batch, each measured in duplicate).
- Sampling method: The samples were filled into 10 mL glass vials. To each vial, 5 mL acetonitrile 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, both additional vessels of NC and A were sampled; one sample per replicate: 2 samples of 5 mL per treatment group.
• After 72 h exposure, both additional vessels and 2 replicates from the test vessels of NC and A were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
For each sampled treatment, one of the analytic samples from 0 h, 24 h, 48 h and 72 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.
- Substance specific analysis was performed at the test site of menal GmbH.
- Concentrations: NC and A
- Sample storage conditions before analysis: Samples were stored at -20°C ± 10°C until analysis, and least until finalization of the phase report after which they will be destroyed or further stored. On assay day, samples were thawed at room temperatures and directly transferred into an HPLC-vial.
- Shipping: Samples were personally handed over between Hydrotox GmbH and menal GmbH

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test was performed with OECD TG 201 medium according to OECD 201 (2006). The stock solution was prepared as Water-Soluble Fraction (WSF) by adding 111.1 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) 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. 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 single test item loading rate in the test (limit test).
Six replicates, with 50 mL test solution (including algal inoculum) each, were prepared for the test item treatment and the negative control.
- Differential loading: No
- Controls: The negative control (NC; test medium) was treated in the same way as the test item solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no chemical vehicle used
- The calculated initial algal biomass in the test vessels was 7 x 10E3 cells/mL

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: Strain No 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettigen
- Age of inoculum (at test initiation): 4-day-old pre-culture was used as inoculum in the test
- Method of cultivation: Twice a week, the stock suspension is inoculated into fresh Holm-Hansen composition: 496 mg/L NaNO3, 39 mg/L K2HPO4, 75 mg/L MgSO4×7H2O, 36 mg/L CaCl2×2H2O, 58 mg/L Na2CO3, 10 mg/L Na2EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2× 4H2O, 0.0088 mg/L ZnSO4×7H2O, 0.0032 mg/L CuSO4×5H2O, 0.0010 mg/L Na2MoO4×2H2O, 0.0016 mg/L CoCl2×6H2O medium under axenic conditions to keep it in exponential growth.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Measurements also taken at 24 hours and 48 hours

Hardness:

Not reported

Test temperature:

22.8 - 22.9 °C

pH:

7.6 - 7.8 (control) and 7.7 - 7.9 (test item treatment)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

0 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL
- Type (delete if not applicable): closed, with cellulose stoppers
- Material, size, headspace, fill volume: test vessels are filled to 50 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no, static test
- Initial cells density: 7 x 10E3 cells/mL
- Control end cells density: 101.994 x 10E5 cells/mL
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 replicates per negative control
- Introduction of algae: The test was started (0 hours) by inoculation of a biomass of approx. 7000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
- Volumes of 50 mL of algal suspension per replicate were rotated at ca. 2 rpm and every 5 rotations the flasks were shaken to minimize differences in light and other test conditions.

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: solutions to make the 10-fold concentrated test medium were dissolved in deionised water; 1-fold concentrated medium was prepared by diluting 10-fold medium with ultrapure water
- Intervals of water quality measurement: The pH was measured at the start and end of the test; vessels were kept in light incubator (21 - 24 °C, constant within ± 2 °C) and continuously illuminated by lateral fluorescent tubes (58 W each), and mean light intensity was reported.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: constant lighting
- Light intensity and quality: Mean light intensity was 71.1 µE m-2 s-1 ± 3.8 %, supplied by lateral fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density, every 24 h; the inhibition of algal growth was determined from yield and growth rate; calculation according to the OECD guideline 201
- Determination of cell concentrations: To convert the measured surrogate parameter chlorophyll fluorescence into a cell concentration (the measure for biomass), a correlation factor is determined twice a year within the quality check by measuring cell concentrations of different dilutions with the Coulter Counter and the corresponding fluorescence with the microplate fluorescence reader. Chlorophyll fluorescence values are correlated with measured cell concentrations. The slope of the curve gives the conversion factor. By means of the conversion factor, the surrogate parameter chlorophyll fluorescence is converted into a cell concentration
- Chlorophyll measurement: The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10 %, the measurement was repeated. Test medium was measured as blank value and subtracted from the fluorescence values in the test vessels.
- Range finding study: Yes, a preliminary test without GLP was performed before start of this GLP-study
- Test concentrations (in range finding study): Nominal loading rate of 100 mg/L test item were tested and resulted in inhibition of -15.8, -5.8 and 20.9% % for yield and -1.7 %, 2.9 % and 4.0 % for growth rate (72 h), respectively.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1. Inhibition of yield after 24 h, 48 h, and 72 h exposure

Nominal test item loading rate [mg/L]	Inhibition of growth rate (%)
	24 h	48 h	72 h
NC	--	--	--
100	-4.4	-9.8	-5.8

Table 2. Inhibition of growth rate after 24 h, 48 h, and 72 h exposure

Nominal test item loading rate [mg/L]	Inhibition of yield (%)
	24 h	48 h	72 h
NC	--	--	--
100	-8.5	-36.8	-30.5

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test according to the OECD guideline 201 Pseudokirchneriella subcapitata was exposed to 0.117 mg/L N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine/L (meas., geom. mean; limit of water solubility; nominal: 100 mg/L) for 72 h. No toxic effect on P. subcapitata was observed.

Executive summary:

In a 72-hour acute toxicity study, cultures of Pseudokirchneriella subcapitata, Strain No. 61.81 SAG were exposed to N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine at a measured concentration of 0.117 mg a.i./L (nominal 100 mg a.i./L) under static conditions in accordance with the OECD guideline 201. The NOEC and LOEC values based on growth rate were ≥ 0.117 and > 0.117 mg a.i./L, respectively. 

The exposure concentration was 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. The geometric mean of the measured test item concentration represents only 0.1 % of the nominal concentration which can be explained by the water solubility of the test item (i.e. below 0.1 mg/L at pH 7.1 and 20 °C in distilled water, see section 4.8 of the present dossier). The limit of water solubility of the test item in test medium was reached. Therefore, N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine is not toxic up to the limit of water solubility to P. subcapitata.

No abnormalities were observed.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

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

 

Effect levels:

(Lowest/No observed) Effect Concentration	Measured test item concentration (mg/L)
	Based on yield (72 hours)	Based on growth rate (72 hours)
LOEC	> 0.117	> 0.117
NOEC	≥ 0.117	≥ 0.117

            

 

Description of key information

The toxicity of N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine was assessed in a 72-hr growth inhibition test with Pseudokirchneriella subcapitata according to OECD test guideline 201 and under GLP. No effects were observed up to the limit of water solubility:

 

NOEC ≥ 0.117 mg a.i./L (measured)

LOEC > 0.117 mg a.i./L (measured)