5'-O-[bis(4-methoxyphenyl)benzyl]-2'-deoxy-N-(2-methyl-1-oxopropyl)guanosine

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:

2017-11-30 to 2018-03-28

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 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 (0 mg/L) and A (100 mg/L nominal) 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.
- Concentrations: NC, 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:

- 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 111.0 mg test item (including a factor of 1.11 to consider the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 48 h using an overhead shaker at 21.4 – 23.0 °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).
- Control: The negative control (NC; test medium) was treated in the same way as the test item solution. - Replicates: Six replicates, with 50 mL test solution (including algal inoculum) each

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 - 23.0 °C

pH:

7.4 - 7.8 (control) and 7.4 - 8.0 (test item treatment)

Dissolved oxygen:

Not reported

Salinity:

Not reported

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 10^3 cells/mL
- Control end cells density: 130.227 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 6 replicates per concentration
- No. of vessels per control (replicates): 6 replicates per negative control

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.

TEST CONCENTRATIONS
- 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 -7.6 %, 2.9 % and 5.9 % 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:

NOEC

Effect conc.:

>= 4.92 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.:

> 4.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

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.

CHEMICAL ANALYSIS

In the control, the test item was not detected. As the measured test item concentrations are not within ± 20% of the nominal loading rates, according to OECD 201 (2006) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations. Therefore, the geometric mean of all four measurement times (0, 24, 48, and 72 hours) was calculated. The achieved concentration is at the limit of water solubility of the test item in test medium. The test item was shaked in test medium for 48 h. Compared to the water solubility in distilled water (i.e. 2.4 mg/L at pH 6.9 and a temperature of 20 °C, see section 4. 8 of this dossier) the solubility of the test item in test item is slightly higher than in distilled water which can be explained by interactions with the salts contained in the medium.

 

CRITERIA OF VALIDITY

- The biomass (cell concentration) in the control has increased by a factor of 88.0 and therefore ≥ 16 during the test period.

- The mean coefficient of variation for section-by-section growth rate (day 0 - 1, 1 - 2 and 2 - 3) in the control is 14.98 % and therefore ≤ 35 %.

- The coefficient of variation of average specific growth rate during the test period in the control replicates is 3.77 % and therefore ≤ 7 %.

- The test is valid according to OECD Test Guideline 201 (23 March 2006).

The percentage of the inhibition of the growth rate is presented in table 1.

Table 1: Inhibition of growth rate after 24, 48 and 72 h exposure

Nominal test item loading range [mg/L]	Inhibition of Growth rate [%]
	24 h	48 h	72 h
NC	--	--	--
100	4.9	-5.8	-6.2

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test according to the OECD guideline 201 Pseudokirchneriella subcapitata was exposed to 4.92 mg/L N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine (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 (limit test), cultures of Pseudokirchneriella subcapitata, Strain No. 61.81 SAG were exposed to N2-Isobutyryl-5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyguanosine at nominal concentration 100 mg test item/L (measured geometric mean. 4.92 mg/L) under static conditions in accordance with the OECD guideline 201. 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. The geometric mean of the measured test item concentration represents only 4.92 % of the nominal concentration which can be explained by the water solubility of the test item (i.e. 2.4 mg/L at pH 6.9 and a temperature of 20 °C, see section 4. 8 of this dossier). The limit of water solubility of the test item in test medium was reached. Therefore, N2-Isobutyryl-5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyguanosine is not toxic up to the limit of water solubility to P. subcapitata.

No abnormalities were observed.

 

This toxicity study 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	> 4.92	> 4.92
NOEC	≥ 4.92	≥ 4.92

Description of key information

The toxicity of N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine was assessed in a 48-hr-acute toxicity test according to OECD test guideline 201 with Pseudokirchneriella subcapitata. No effects were observed up to the limit of water solubility:

 

The following effect levels - based on the growth rate - are calculated:

- 72 h LOEC > 4.92 mg a.i./L

- 72 h NOEC ≥ 4.92 mg a.i./L

Additional information

The 72-hr-acute toxicity of N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine to Pseudokirchneriella subcapitata was studied under static conditions. Algae were exposed to control, and test chemical at measured concentration of 4.92 mg/L for 72-hr in a limit test (nominal loading rate 100 mg/L).  Based on the inhibition of growth rate the 72-hr NOEC equals or is above 4.92 mg/L.

 

The initial concentration and the maintenance of the exposure concentrations during the test were verified in the analytical part.

The achieved concentration is at the limit of the water solubility of the test item in test medium. The test item was shaked in test medium for 48 h. Compared to the water solubility in distilled water (i.e. 2.4 mg/L at pH 6.9 and a temperature of 20 °C, see section 4. 8 of this dossier) the solubility of the test item in test medium is slightly higher than in distilled water which can be explained by interactions with the salts contained in the medium.

 

The No Observed Effect Concentration (NOEC) is ≥ 4.92 mg/L and the Lowest Observed Effect Concentration (LOEC) is > 4.92 mg/L at 72 hours.

  

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