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

Long-term toxicity to fish

Administrative data

Endpoint:

adult fish: sub(lethal) effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-23 to 2014-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant and based on OECD test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

White to light yellow crystalline powder. Negligible solubility in water.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For analysis of the actual test substance concentration, 2 ml of test solution was taken from each test vessel at the beginning, before and after each renewal and at the end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

500 µL of test samples, test water spiked samples and test water blank were analyzed by UPLC after adding 1-fold dilution methanol as solubilising agent to make sample dissolve completely.

Test organisms

Test organisms (species):

other: Chinese Rare Minnow (Gobiocypris rarus)

Details on test organisms:

The test was performed with Gobiocypris rarus which were bred in Bioassay and Safety Assessment Laboratory of Shanghai Academy of Public Measurement (ID: GR-SAPM-130506). This freshwater fish species is suitable for fish toxicity testing because of their ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species. This species complies with OECD Guidelines for the Testing of Chemicals. 204 Fish, Prolonged Toxicity Test: 14-day Study 1984.

Before being used in the test, all fish were acclimatized in the following test conditions for more than 14 days:
Photoperiod: Light : Dark=12 hours : 12 hours;

Feeding: fish were fed with Brine Shrimp Aquarium Flake Food, O.S.I® daily
The criterion of fish selection:
Length was 2.1 ± 0.1cm (Mean± SD). body weight was 0.16 ± 0.02g (Mean± SD) at the beginning of the test. Length was 2.2 ± 0.1 cm (Mean± SD), body weight was 0.18 ± 0.02g (Mean ± SD) at the end of the test.

No diseases were observed during the acclimating period;

No mortality was present within 7 days before test beginning

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

14 d

Remarks on exposure duration:

The fish were inspected and recorded once a day for mortality and other observed effects. Study duration: 14 days.

Test conditions

Hardness:

Total hardness: 155 mg CaC03 per liter

Test temperature:

21-25°C

pH:

6.0-8.5

Dissolved oxygen:

> 80% of air saturation value (ASV)

Nominal and measured concentrations:

A nominal concentration of 100 mg/L of the test substance was prepared.
UPLC showed that, the measured concentrations of the test substance in the test solutions were all below LOD21(LOD2=0.04mg/L).

Details on test conditions:

Test conditions
Water:
The tap water dechlorinated by activated carbon and sterilized by UV lamp was used. The water was aerated continuously over 24h. Water quality parameters were as following:
Total hardness: 155 mg CaC03 per liter;
Oxygen concentration: >90% of ASV.

Test vessels
5L glass tanks were used containing 4L test solution.

Conditions of exposure
Duration: 14d;
Test method: Semi-static and the frequency of renewal was twice a week;
Loading: about 0.41-0.46 fish/litre;
Temperature: 21.6°C-23.4 ·c;
Photoperiod: Light : Dark= 12 hours : 12 hours;
Oxygen concentration: 73.3%-95.0% ASV;
Feeding: fish were fed twice a day and the quantity of food being kept constant. The amount of food was at a level of approximately 1 % of body weight per day
Cleaning: the test vessel was replaced by a clean one each time the test solution was renewed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

>= 0.04 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: saturated solution

Effect concentrations 2

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.04 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: saturated solution

Details on results:

The actual test substance concentration of test solutions was determined by using a UPLC analysis method. The limit of detection (LOD2) for the test substance was calculated to be 0.04 mg/L (based on a signal/noise of 6.46).The limit of quantification (LOQ2) for the test substance was calculated to be 1 mg/L (based on a signal/noise of 15.4). It was found that the average recoveries were 101 % and 94.9% of the spiked 0.60 mg/Land 6.00 mg/L sample with RSD value 1.71 % and 4.40%, respectively. During the test, the measured concentrations of the test substance in test solutions were all below the LOD2

There were no mortalities and abnormalities observed in treatment group during the test. At the end of the test, the body weight of the test fish increased in both groups. The specific growth rate of the body weight in treatment group was not significantly different from that of the control (p>0.05)

Results with reference substance (positive control):

No reference substance used in the study.

Reported statistics and error estimates:

To determine the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC), the variances among the mortality and the specific growth rate in the treatment group was analyzed by Shapiro-Wilk's Test and F-Test and compared with the control group using Wilcoxon Two-Sample test. The data treatment was carried out with software TOXCALC.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

For the lethal effect and the growth effect , both the the median lethal concentration (LC50)and the lowest observed effect concentration (LOEC) for Gobiocypris rarus exposed to the test substance Proketal (NJBY20130218-0718) in 14d were higher than the concentration of the saturated solution (measured concentrations were