(2S-cis)-(diphenylmethyl)-N-(phenylmethyl)-1-azabicyclo[2.2.2.]octan-3-amine (1-R camphor-10-sulfonate)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-810-6

EC Name:

-

Molecular formula:

C27 H30 N2 * C10 H16 O4 S

IUPAC Name:

(2S-cis)-(diphenylmethyl)-N-(phenylmethyl)-1-azabicyclo[2.2.2.]octan-3-amine (1-R camphor-10-sulfonate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: CP-163,625-BV
Batch (Lot) Number: INT 18 320
Expiry date: 16 April 2020 (retest date)
Physical Description: White powder
Purity/Composition: 97.6%
Storage Conditions: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Solutions containing 4.6, 10, 22, 46 and 100% of the Saturated solution, prepared at a loading rate of 100 mg/L.
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis.
Frequency at t=0 h and t=48 h
Volume 2.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of CP-163,625-BV tested was a white powder with a purity of 97.8% which was visually not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a 15 minute period of ultrasonic treatment followed by three days of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source In-house laboratory culture with a known history.
Reason for selection This system has been selected as an internationally accepted invertebrate species.
Validity of batch Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured
animals and there was no delay in the production of the first brood.
Characteristics Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding
Start of each batch Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all glass culture vessel.
Maximum age of cultures 4 weeks
Renewal of the cultures After 7 days of cultivation, half of the medium twice a week.
Temperature of medium 18-22°C
Feeding Daily, a suspension of fresh water algae.
Culture medium M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18 22°C, constant within ±1°C).

pH:

These test conditions remained within the limits prescribed by the study plan (pH: 6- 9, not varying by more than 1.5 units)

Dissolved oxygen:

These test conditions remained within the limits prescribed by the study plan ( oxygen: >=3 mg/L at the end of the test).

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal : Solutions containing 4.6, 10, 22, 46 and 100% of the Saturated Solution, prepared at a loading rate of 100 mg/L.
Measured: Despite filtration of the SS during preparation of test solutions, the analysed concentration in the SS was at the level of the nominal loading rate. Taking the dilution factor applied, dilutions of the SS were equivalent to nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Material, size, headspace, fill volume: glass, 60 mL, 10 mL, 50 mL
- Volume of solution: 50mL
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): four
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): four


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

- Culture medium different from test medium: yes
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: N/A
- Photoperiod: 16 hours
- Light intensity: not given

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality) At 24 hours and at 48 hours.
pH and dissolved oxygen At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium Continuously in a temperature control vessel, beginning at the start of the test.

VEHICLE CONTROL PERFORMED:N/A

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100% of saturated solution
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 57 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 27 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Results with reference substance (positive control):

The study met the acceptability criteria prescribed by the study plan and was considered valid.
The 24h-EC50 in this reference test with K2Cr2O7 was within the expected range of 0.6 and 2.1 mg/L as specified in ISO International Standard 6341, October 2012. The 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
The 24h-EC50 was 0.92 mg/L with a 95% confidence interval between 0.78 and 1.1 mg/L.
The 48h-EC50 was 0.55 mg/L with a 95% confidence interval between 0.47 and 0.64 mg/L.
In conclusion, the sensitivity of this batch of Daphnia magna was in agreement with ISO International Standard 6341, October 2012 and the historical data collected at Charles River Den Bosch.

Reported statistics and error estimates:

24 hr: Value [mg/L] 57.218
lower 95%-cl 40.781
upper 95%-cl 84.785

48 hr:Parameter EC50
Value [mg/L] 27.180
lower 95%-cl 21.201
upper 95%-cl 35.892

Any other information on results incl. tables

 Combined Limit/Range-Finding Test

Table 1shows the responses recorded during the combined limit/range-finding test.

No immobility was observed in the control and solution containing 1.0% of the SS throughout the test. At the end of the test, 40 and 100% immobility was observed in solutions containing 10 and 100% of the SS, respectively.

Based on these results, samples taken from solutions containing 1.0 and 100% of the SS were analysed. The measured concentrations at the start of the test were 0.74 and 72 mg/L, respectively. The concentrations remained stable throughout the exposure period, i.e. were at 92 and 97% relative to the initial concentrations at the end of the test (see also Table 2 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Table 1          
Number of Introduced Daphnids and Incidence of Immobility in the Combined Limit/Range-Finding Test

 

Time (h)	Replicate	CP-163,625-BV;%SS prep. at a loading rate of 100 mg/L
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	4^#
	B	0	0	0	3*
	C	0			3*
	D	0			3*
	Total immobilised	0	0	0	13
	Effect %	0	0	0	65
48	A	0	0	2	5
	B	0	0	2	5*
	C	0			5*
	D	0			5*
	Total immobilised	0	0	4	20
	Effect %	0	0	40	100

^#Microscopic observation revealed no test item was attached to the daphnids.

* A floating layer of undissolved material was observed in this replicate.

Final Test

Measured Concentrations

The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report.

Samples taken from all test concentrations and the control were analysed. Despite filtration of the SS during preparation of test solutions, the analysed concentration in the SS was at the level of the nominal loading rate, i.e. remained stable at 100 mg/L throughout the test. Hence, it can be concluded that the undissolved material removed during preparation of test solutions was not test item related. Taking the dilution factor applied, dilutions of the SS were equivalent to nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L.

The measured concentrations were in the range of 96-101% relative to nominal concentrations throughout the test. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

It should be noted that small test item responses were detected in the control samples, which most likely originated from carry-over in the analytical instrument since the similar was observed in the blank Quality Control (QC) samples and analytical blanks.

Immobility

Table 2 shows the responses recorded during the final test.

No immobility was observed in the control and at the lowest test concentration throughout the test. A concentration-related increase of immobility was observed at nominally 10 mg/L and higher, resulting in 70 and 100% immobility at the highest test concentration after 24 and 48 hours of exposure, respectively.

The responses recorded in this test allowed for reliable determination of EC₅₀-values and responses were comparable to those recorded in the combined limit/range-finding test.

Table 2          
Numberof Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)	Replicate	CP-163,625-BV; Nominal conc. (mg/L)
		Control	4.6	10	22	46	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	2	1	3
	B	0	0	0	2	1	3
	C	0	0	0	0	3	4
	D	0	0	1	3^#	3	4
	Total immobilised	0	0	1	7	8	14
	Effect %	0	0	5	35	40	70
48	A	0	0	1	4	3	5
	B	0	0	1	2	2	5
	C	0	0	1	2	5	5
	D	0	0	2	3	2	5
	Total immobilised	0	0	5	11	12	20
	Effect %	0	0	25	55	60	100

^#Microscopic observation revealed no test item attached to the daphnids.

 Experimental Conditions

The results of measurement of pH and oxygen concentrations (mg/L) are presented inTable 4. These test conditions remained within the limits prescribed by the study plan (pH: 6‑9, not varying by more than 1.5 units; oxygen: >=3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18‑22°C, constant within±1°C).

Table 4          
pH and Oxygen Concentrations (mg/L) during the Final Test

CP-163,625-BV; Nominal conc. (mg/L)	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	9.4	7.6	9.3	8.2
4.6	9.4	7.6	9.2	8.2
10	9.3	7.6	9.2	8.2
22	9.2	7.6	9.2	8.2
46	9.1	7.6	9.2	8.1
100	8.7	7.5	9.1	8.1

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to CP-163,625-BV was 27 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 21 and 36 mg/L).

Executive summary:

The objective of the study was to evaluate CP-163,625-BV for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC₅₀ at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23,2019.

The batch of CP-163,625-BV tested was a white powder with a purity of 97.8% which was visually not completely soluble in test medium at the loading rate initially prepared.A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. Despite filtration of the SS during preparation of test solutions, the analysed concentration in the SS was at the level of the nominal loading rate, i.e. remained stable at 100 mg/L throughout the test. Taking the dilution factor applied, dilutions of the SS were equivalent to nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L. The measured concentrations were in the range of 95-101% relative to nominal concentrations throughout the test. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

No immobility was observed in the control and at the lowest test concentration throughout the test. A concentration-related increase of immobility was observed at nominally 10 mg/L and higher, resulting in 70 and 100% immobility at the highest test concentration after 24 and 48 hours of exposure, respectively.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to CP-163,625-BV was 27 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 21 and 36 mg/L).