Dodecahydro-3,8,8,11a-tetramethyl-5H-3,5a-epoxynaphth[2,1-c]oxepin

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 28, 2017 to November 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

see "Principles of method if other than guideline" field below

Principles of method if other than guideline:

A closed-bottle test system was used to reduce losses of volatile components of the test item. Thus, the test medium was supplemented with sodium bicarbonate to increase the CO2 concentration in the test medium to achieve maximum algal growth rates without pH drift (Mayer et al. (2000), ISO 14442 (2006)).
Deviating from the study plan the pH in the test medium exceeded the required upper limit (pH = 9) by 0.4 pH units. This deviation has no influence on the integrity of the study results, since all cell growth related validity criteria were fulfilled and the pH drift throughout all well growing treatment levels were always equal to 1.0 pH units. Otherwise, the study was performed according to the guideline, the study plan and its amendment(s).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

rel-(3S,5aR,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Molecular formula:

C18H30O2

IUPAC Name:

rel-(3S,5aR,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Constituent 2

Reference substance name:

rel-(3R,5aS,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Cas Number:

220432-47-3

Molecular formula:

C18H30O2

IUPAC Name:

rel-(3R,5aS,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.: VE00484383
- Purity: 99.8% (major peak)
- Stability under correct storage conditions (expiry date): August 23, 2017

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Samples were analysed at the start of exposure (0h) and at the end of exposure (72h).
Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded. After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of 5 -18°C if not advised otherwise. A record was kept for each sample.

Test solutions

Vehicle:

no

Details on test solutions:

The following approach was used taking into account aspects of ISO 14442 (2006), Water quality - Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water, and OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment Number 23. ENV/JM/MON0(2000)6.
A saturated stock solution was prepared by suspending the test item in test medium. A stock solution at a nominal loading rate of 100 mg/L was prepared by directly adding the test item to the test medium and slowly stirring for 142 hours at ambient temperature in the dark. Thereafter the stock solution was left to settle for =>0.5 hour. The stock solution was visually examined for undissolved/particulate matter. Particles of the test item were visible, so the solution was filtered using a glass fibre filter (MN GF-5) with an average retention capacity of 0.45 pm. The initial 0.25 L of the filtrate was discarded. The filtrate was visually examined for undissolved/particulate matter by Tyndall-effect and no undissolved/particulate matter were visible. The filtrate was used directly (100% treatment) and diluted to prepare the desired test item concentration.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test system used in this study was the freshwater algae Pseudokirchneriella subcapitata (SAG 61.81).
The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21-24 °C, controlled at ±2 °C

pH:

8.4 - 9.4 in test solution

Nominal and measured concentrations:

Nominal concentration: 100, 66.7, 44.5, 29.7, 19.8% of a saturated solution at a loading rate of 100 mg/L.
Measured concentrations (test period: 0 h) : 1.120, 0.807, 0.527, 0.350 and 0.317 mg/L of AMBERKETAL
Measured concentrations (test period: 72 h) : 1.020, 0.666, 0.392, 0.221 and 0.201 mg/L of AMBERKETAL
Measured concentrations (geometric mean): 1.07, 0.733, 0.455, 0.278 and 0.252 mg/L of AMBERKETAL

Details on test conditions:

- Test vessels: 60 mL glass vials, air-tightly closed by a screw-cap with PTFE lining
- Volume of test solution per test vessel: 60±2 mL
- Age of the pre-culture: 4 days
- Number of cells per mL in the pre-culture before inoculating the test solutions: 264x104
- Number of cells per mL test solution at the beginning of the test: 0.3x104
- Number of replicates per test item concentration: 3
- Number of replicates in the control: 6
- Number of replicates for stability check (intermediate and highest concentration level without algae): 2 (without algae)
- Test medium: OECD medium (modified) + additional NaHCO3
- pH of test medium: 8.3
- pH in test solutions: 8.4-9.4; for details see section 18.1 and 17
- Light regime: 24 h light/0 h dark
- Type of light: Econlux LED Sunstrip "daylight"
- Light intensity: 70.2-78.5 pE rrr2S-1;mean 74.7 pE M-2S-1;
- Temperature: 22.1-22.6, mean 22.33 °C; for details see
- Shaker: 100±5 oscillations/min; the test vessels were placed randomly on a horizontal shaker, so that each test vessel was rotated around its own axis
- Test duration (exposure): 72 hours
- Counting of algae: Daily

Reference substance (positive control):

yes

Remarks:

Two parallel reference tests using potassium dichromate (K2Cr207) as reference item were performed in April 2017 in separate studies

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

saturated solution at a loading rate of 100 mg test item/L.

Basis for effect:

growth rate

Remarks on result:

other: < 10% inhibition seen at highest test concentration (100% saturated solution)

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

saturated solution at a loading rate of 100 mg test item/L.

Basis for effect:

growth rate

Remarks on result:

other: < 10% inhibition seen at highest test concentration (100% Saturated Solution)

Details on results:

A weak concentration-response relationship was observed for the biological parameter yield during the exposure period, and no concentration-response relationship was observed for the biological parameter growth rate. The % inhibition observed in the five test concentrations was:

For the parameter yield: 0% (Control), 0.8% (0.252 mg/L), 1.6% (0.278 mg/L), 9.0% (0.455 mg/L), 3.1% (0.733 mg/L) and 15.9% (1.07 mg/L)
For the parameter growth rate: 0% (Control), 0.1% (0.252 mg/L), 0.3% (0.278 mg/L), 1.8% (0.455 mg/L), 0.6% (0.733 mg/L) and 3.4% (1.07 mg/L)

The %-inhibition seen for the parameter yield was <20%. Therefore, the EC20, and EC50 were stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 1.07 mg Amberketal/L). The NOEC and LOEC were statistically determined to be 0.733 mg/L and 1.07 mg/L.

The %-inhibition seen for the parameter growth rate was <10%. Therefore, the EC10, EC20 and EC50 were stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 1.07 mg Amberketal/L). The NOEC and LOEC were statistically determined to be 0.733 mg/L and 11.07 mg/L. However, the 3.4% inhibition seen at the highest concentration is not considered to be biologically significant by the lead registrant, in particular as a closed system was used which tends to put the algae under a little more stress than tests performed under a conventional set up (see "Result with reference substance" below).

The preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The OECD201 guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. It is also noted that toxicity values calculated by using these 2 response variables are not comparable. ECx values based upon average specific growth rate (ErCx) will generally be higher than results based upon yield (EyCx) if the test conditions of this guideline are adhered to, due to the mathematical bases of the respective approaches. This should not be interpreted as a difference in sensitivity between the 2 response variables, simply that the values are different mathematically. The ECHA guidance explains that effects based on biomass should not be used because " direct use of the biomass concentration without logarithmic transformation cannot be applied to an analysis of results from a system in exponential growth". The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table. Furthermore, the preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus, only the EC10 is presented in the above "effects concentration" table.

Results with reference substance (positive control):

The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.860 mg/L (standard system) and 0.753 mg/L (closed system). These values are within the historical range of the general reference test results (standard open test system) of the test laboratory (mean ErC50 value = 1.072 ± 0.357 mg/L).

Any other information on results incl. tables

Samples from the test solutions were analysed to determine actual levels of the test item at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours. The latter were 63-91% of the initial measured concentrations. Thus, the test item concentration based on initial measured concentration remained not stable within ±20% throughout the exposure period. Since all concentration levels were measured, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled as required by the study plan.

Conclusions:

The ErC10 and ErC50 Amberketal of Pseudokirchneriella subcapitata was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 1.07 mg Amberketal/L). Thus based on growth rate, Amberketal was considered to exhibit no biologically significant effect up to its solubility limit.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Amberketal was determined according to OECD guideline No. 201.

The Batch of AMBERKETAL tested was a whitish to pale yellow solid.

As the test item is poorly water-soluble, the following approach was used taking into account aspects of OECD 2000, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment Number 23. ENV/JM/MONO(2000)6. A saturated stock solution was prepared by suspending the test item in test medium. A stock solution at a nominal loading rate of 100 mg/L was prepared by directly adding the test item to the test medium. and slowly stirring for 142 hours at ambient temperature in the dark. Thereafter the stock solution was left to settle for =>0.5 hour. The stock solution was visually examined for undissolved/particulate matter. Particles of the test item were visible, so the solution was filtered using a glass fibre filter (MN GF-5) with an average retention capacity of 0.45 pm. The initial 0.25 L of the filtrate was discarded. The filtrate was visually examined for undissolved/particulate matter by Tyndall-effect and no undissolved/particulate matter were visible. The filtrate was used directly (100% treatment) and diluted to prepare the desired test item concentration.

The following concentration steps in a geometrical series (spacing factor: 1.5) were tested in the definitive test: 100, 66.7, 44.5, 29.7, 19.8% of a saturated solution at a loading rate of 100 mg/L. Additionally, the test organisms were exposed under control conditions (non-treated test medium). The test item was applied once at the beginning of the exposure period. Three replicate vessels were used per test item concentration, and six replicate vessels for the control. Additional test vessels without algae were prepared for potential chemical analysis of the intermediate and the highest test item concentration at the end of the test, in order to assess test item stability and potential algal uptake during exposure without the presence of algae (stability samples). The additional vessels were maintained under the same test conditions as the vessels with algae, with the exceptions described above. The test period (exposure of test organisms to the test solutions in a static system) was 72 hours.

Samples from the test solutions were analysed to determine actual levels of the test item at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours. The latter were 63-91% of the initial measured concentrations. Thus, the test item concentration based on initial measured concentration remained not stable within ±20% throughout the exposure

period. Since all concentration levels were measured, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level.

A weak concentration-response relationship was observed for the biological parameter yield during the exposure period, and no concentration-response relationship was observed for the biological parameter growth rate.

The %-inhibition seen for the parameter growth rate was <10%. Therefore, the ErC10 and ErC50 were stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 1.07 mg Amberketal/L).