3,20-bis(ethylenedioxy)pregna-5,7-diene

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-03-06 to 1998-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A GLP study conducted to current accepted testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Yes, (incl Certificate) OECD Good Laboratory Principles and Compliance Monitoring N0. 1 ENV/MC/CHEM(98) 1998

Test material

Test material information

Constituent 1

Reference substance name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

EC Number:

243-175-0

EC Name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

Cas Number:

19592-55-3

Molecular formula:

C25H36O4

IUPAC Name:

(1S,3aR,9aR,9bS,11aS)-9a,11a-dimethyl-1-(2-methyl-1,3-dioxolan-2-yl)-1,2,3,3a,6,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolane]

Test material form:

solid: crystalline

Details on test material:

Appearance: white crystalline powder

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Purity: 99.0% (NMR}

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100mg/l

- Sampling method: On day 0, one sample of 25ml was taken of all beakers and on day 4 one sample of 25ml was taken of the glass beakers filled with test solution. Immediately after sampling 3ml of methanol was added.
All samples were extracted by shaking 3 times with 25ml of dichloromethane. The Dichloromethane fractions were transferred to a Turbovap tube (75ml) and evaporated with the Turbovap at 40C util 1ml (= sensor endpoint) The residue was dissolved in 5ml dichloromethane by shaking and ultrasonic treatment and transferred into a test tube. Again 2ml acetonitrile was added to dissolve the residue by shaking and ultrasonic treatment and transferred into the same test tube. The 8ml was evaporated with the Turbovap at 40C. The residue was dissolved in 5.0ml mobile phase by one minute ultrasonic treatment and after transfer into a HPLC vial, analysed by HPLC.

- Sample storage conditions before analysis: All samples were stored at a temperature between 2 and 10C until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The final study was conducted with saturated solutions in ISO-water. Two glass beakers were prepared per test concentration (151.1 and 150.3 mg in 1.5 L ISO water). Each beaker contained 1.5 L of test solution and 7 zebra fish.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): following stirring for 24.5 hrs. Both beakers were filtered to reach solubility level. Particles of the test substance were not visible in both test solutions.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Zebra fish (Brachydanio rerio) used in these tests originated from a commercial supplier (Bio International, Horn, The Netherlands). The fish arrived at the laboratory of the Environmental Research Department, Solvay Pharmaceuticals on 13 January 1998. The fish were kept in climate chamber A105c in a plastic container with 80 litre ISO-water.
The setpoint of the temperature in the climate chamber was 23°C. The light regime was 14 hours light and 10 hours dark (light on from 8 am
to 10 pm). The fish were fed with Tetramin® and living water fleas. The water was filtered continuously during the acclimation period to assure an
acceptable water quality.

No aberrant behaviour or appearance of the fish was observed at the day of test initiation. The total
weight of 30 fish was determined at the day of test initiation. The total weight of the 30 fish was 2.23 gram which results in a mean weight of 0.074 gram per fish so the mean weight per liter test solution is< 1 g/l as stated in the protocol. These fish were returned to the plastic container before test initiation. The fish were not fed during the 24-hour period prior to the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Three, 24, 48, 72 and 96 hours after the start of the test, the behaviour and general appearance of each fish was recorded. At the end of the test the surviving fish were killed with Hypnodil. The total length (tail included) of each dead fish was determined. The control fish of the test were not killed with Hypnodil but were used for further studies

Test conditions

Test temperature:

The temperature in the solutions ranged between 22.4 and 24.4C

pH:

The pH of the solutions ranged between 8.0 and 8.2 during the test

Dissolved oxygen:

The measured oxygen content of the test solutions ranged between 8.0 and 8.5mg/l during the test

Nominal and measured concentrations:

The study was conducted at nominal concentrations of 0

Results of HPLC analysis revealed the following measured concentration HPLC sample values (µg/l):
Proketal A 0h. (solubility level): 553
Proketal B 0h. (solubility level): 485
Proketal A 96h. (solubility level): 66.1
Proketal B 96 h. (solubility level): 71.9

Details on test conditions:

The study was conducted in climate chamber A105a. The setpoint of the temperature in the climate chamber was 23.0°C. The air temperature in the climate chamber was recorded continuously during the test and this measured air temperature ranged between 22 and 23 °C during the test. The light regime was 14 hours light and 10 hours dark (light on from 8 am to 10 pm). The zebra fish were not fed during the test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No mortality was observed in the control glass beakers. Because neither mortality nor effects on behaviour and general appearance were observed in the saturated solutions that originated from a suspension of 100 mg/I, this concentration is considered a No Observed Effect Concentration (NOEC

Any other information on results incl. tables

Sublethal observations / clinical signs:

To study the exposure of zebra fish to proketal, samples of the test solutions were taken and analysed by HPLC. Samples were taken on days 0 and 4. After correction for recovery the mean concentration during exposure of each glass beaker decreased from 800 and 710 µg/1 on day 0 to 96.1 and 105 µg/1 after 4 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No mortality was observed in the control glass beakers. Because neither mortality nor effects on behaviour and general appearance were observed in the saturated solutions that originated from a suspension of 100 mg/I, this concentration is considered a No Observed Effect Concentration (NOEC).