N-benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine

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:

2017-12-06 to 2018-03-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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: Three sampling time points at each of 6 test concentrations (18 samples total).
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, all replicates of NC and A were sampled; one sample per replicate: 2 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
- Storage: All samples were stored at menal GmbH 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 Fractions (WSF) by adding 400.2 mg test item to 500 mL test medium and shaking for 48 h using an overhead shaker at 21.2 – 23.0 °C in the dark. The WSF was filtered through a fibre-glass filter with a retaining range up to 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 concentration 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
- Origin: 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 withEC50 (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:

evaluation after 24 and 48 hours

Hardness:

not reported

Test temperature:

19.9-21.0 °C

pH:

7.5-8.2 (control); 7.6-8.3 (test item)

Dissolved oxygen:

8.0-8.4 mg/L (control); 8.0-8.3 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

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: Nominal loading rates of 10, 100 and 200 mg/L test item were tested and resulted in 0, 0 and 20 % 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.:

0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: value equals the LOD

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

<= 0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: value equals the LOD

Details on results:

- Behavioural abnormalities: Besides immobility no abnormalities are reported
- Mortality of control: no mortality
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- 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.
Depending on the sensitivity of the available analytical method at the test site, the concentrations detected were below the LOQ (= 1 mg/L). Some concentrations were determined above the LOD (= 0.05 mg/L) and the LOQ (see table 1). Those values were considered for determination of ECx values. For nominal loadings within which the concentrations were below the LOD, the LOD was used for determination of the concentration response curve.

- ANALYTICAL RESULTS:

As the measured test item concentrations are not within ± 20 % of the nominal loading rates, 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. For measurements which are < LOQ, and did not result in a measurement value, the value of the limit of detection (LOD), i.e. 0.05 mg/L, was used for calculation.

The chemical analysis was conducted using the method with the lowest LOQ possible (LOQ = 1 mg/L) 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). See Table 1.

Table 1. Analytically measured test item concentrations after 0 h, 24, h, and 48 h of exposure (LOQ = 1 mg/L, LOD = 0.05 mg/L); the values in brackets indicate measurements below the LOQ and above the LOD.

Nominal test item loading rate (mg/L)	Sample	Sampling time (h)	Measured test item concentration (mg/L)	Geometric mean test item concentration based on mesured values below the LOQ and above the LOD (mg/L)
NC	NC1	0	< LOQ	--
NC	NC2	24	< LOQ	--
NC	NC3	48	< LOQ	--
25	A1	0	< LOQ	0.05
25	A2	24	< LOQ	0.05
25	A3	48	< LOQ	0.05
50	B1	0	< LOQ	0.05
50	B2	24	< LOQ	0.05
50	B3	48	< LOQ	0.05
100	C1	0	< LOQ	0.05
100	C2	24	< LOQ	0.05
100	C3	48	< LOQ	0.05
200	D1	0	< LOQ (0.279)	0.210
200	D2	24	< LOQ (0.192)	0.210
200	D3	48	< LOQ (0.172)	0.210
400	E1	0	< LOQ (0.527)	0.190
400	E2	24	< LOQ (0.259)	0.190
400	E3	48	< LOQ *	0.190

*The LOD was used for calculation of the geometric mean.

In the highest concentration the test substance could not be quantified after 48 h (see table 1). Therefore, the measurement value for this point in time was set to the LOD (0.05 mg/L). The EC50 based on mean measured concentrations was estimated at 0.17 mg/L. The underlying dose-response-curve for 48 h exposure is not fully plausible in view of the apparently declining measured concentrations (Table 2). Nevertheless, the presented EC50 value of 0.17 mg/L is conservative (being below the concentration of observed EC30) and therefore relevant for the hazard assessment. In any case, the data support an EC50 of < 1 mg/L which is the relevant key information for classification.

- 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	0.05	0	0
50	0.05	0	0
100	0.05	0	0
200	0.210	30	30
400	0.190	100	100

- 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 ≥ 8.0 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 0.17 mg/L (meas., geom. mean) was determined for N-Benzoyl-5'O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine.

Executive summary:

The 48-hr-acute toxicity of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine to Daphnia magna was studied under static conditions. Daphnids were exposed to control, and test chemical at nominal concentrations of 25, 50, 100, 200, and 400 mg/L for 48 hours. The chemical analysis for the acute toxicity study with D. magna was conducted using the method with the lowest LOQ possible (LOQ = 1 mg/L) 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). All measured concentrations were below the LOQ. Some concentrations were determined above the LOD (= 0.05 mg/L) and the LOQ (see table 1). Those values were considered for calculation of ECx values. For test solutions within which the concentrations were below the LOD, the LOD was used for determination of the concentration response curve.

The 48-hour EC50 based on these values was 0.170 mg a.i./L. Mortality, immobilization and sublethal effects were observed daily.

According to the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint specific guidance, Version 4.0, June 2017 (Table R.7.8-3) if toxicity is observed below the detection limit of the analytical method, “the lowest effect concentration may be based on either the water solubility limit or detection limit of the analytical method, whichever is the lower.” This strategy is considered as a realistic worst case. Therefore, the LOEC is set as the defined LOD of 0.05 mg/L (the water solubility limit is determined as 0.14 mg/L, see endpoint 4.8 in the present dossier) and the NOEC equals or is below the LOD, as no effects were seen in the test solutions containing the test item in a concentration equalling or below the LOD (see table 2). The presented EC50 value of 0.170 mg a.i./L is conservative (being below the concentration of observed EC30) and therefore relevant for the hazard assessment. In any case, the data support an EC50 of < 1 mg/L which is the relevant key information for classification.

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

The study complies with classification requirements, as the analytic is sensitive enough to substantiate that effects on D. magna were seen below the threshold of classification for "Aquatic Acute 1" (H400) and “Aquatic Chronic 1 (H410)”.

 

Results synopsis

 

Test organism age (e.g. 1st instar): < 24 h

Test type: Static

 

NOEC ≤ 0.05 mg a.i./L (based on LOD)

LOEC ≥ 0.05 mg a.i./L (based on LOD)

EC50 = 0.170 mg a.i./L (meas. geom. mean)

 

Endpoint(s) effected: Mobility

Description of key information

The toxicity of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine 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:

 

LOEC ≥ 0.05 mg/L ( = LOD)

NOEC ≤ 0.05 mg/L ( ≤ LOD)

EC50 = 0.170 mg a.i./L (measured)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.17 mg/L