2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-5-yl)propanoyl]amino]-2-methyl-propanoic acid Trifluoroacetic acid salt (1:1)

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

Study period:

START OF EXPERIMENT: 17 February 2021 - END OF EXPERIMENT: 19 February 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(1H-imidazol-5-yl)propanamido]-2-methylpropanoic acid; trifluoroacetic acid

EC Number:

815-122-7

Cas Number:

1446013-08-6

Molecular formula:

C27H27F3N4O7

IUPAC Name:

2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(1H-imidazol-5-yl)propanamido]-2-methylpropanoic acid; trifluoroacetic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: FMOC HIS-AIB-OH TFA LS
CAS number: 1446013-08-6
Batch/Lot number: 0020002355
Description: White powder
Purity: 97-100 % (treated as 100%)
Manufacturing date: 13 September 2020
Expiry date: 23 August 2024
Storage conditions: Refrigerated (2-8 ºC), tight closed container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The method for sample preparation and analysis was developed by the Test Facility. The detailed description of the analytical method is presented in the report on the analytical method validation (Study code: N20017-917). Samples taken at the start as well as at the end of the daphnia test were immediately transported to the Test Facility for analysis. During transportation, the samples were refrigerated.
Sample preparation method
Samples at the START of the daphnia limit test:
approximately 10 mL sample from both the control and from the saturated test item solution was provided by the Test Site in centrifuge tubes for analysis. The solutions were properly diluted prior to HPLC analysis:
- control: 1:1 dilution was prepared with acetonitrile in duplicates
- test item stock solution: 20 μL acetonitrile and 960 μL diluent were added to 20 μL sample, then this solution was further diluted by 2 fold with the diluent (100 fold dilution in total); dilutions were made in 5 replicates
Samples at the END of the daphnia limit test: approximately 10 mL sample from each of the
6 control flasks and 7 test flasks were provided by the Test Site for analysis. The solutions
were properly diluted prior to HPLC analysis:
- control 1 solution: 1:1 dilution was prepared with acetonitrile in duplicates
- test item stock solutions 1-7: 50 μL solution was diluted to 5 mL with the diluent.
Two dilutions were made from each solution.

Test solutions

Details on test solutions:

Preparation of Testing Solutions
Due to the moderate solubility of the test item, oversaturated solutions were prepared in the daphnia media using 400 mg/L test item. Dissolution was assured by sonication for 30 minutes. The oversaturated solutions were filtered through a 0.22 µm cellulose acetate filter. As a result of the filtration, water clear stock solutions were obtained. The test solutions were prepared just before introduction to the daphnids (start of the experiment).
Preliminary Range-Finding Test
A Preliminary Range-Finding test was conducted to determine the approximate toxicity (if any) of the test item, so that appropriate test concentrations can be selected for use in the main test. Thirty daphnids per concentration divided into three groups were exposed for 48 hours.
A stock solution of 400 mg/L (based on the nominal concentration) was first prepared by dissolving the test item in tap water. The test solutions were prepared by appropriate dilution (10, 100, 1000 and 10000 fold) of this stock solution.
No significant toxic effects were observed during the preliminary range-finding test, therefore only one test concentration (nominal 400 mg/L) and one control group were tested in a limit test.
Limit Test
Based on the results of the range-finding test – where no adverse effect was observed – a limit test was conducted in order to demonstrate that the LC50 is greater than the maximum concentration applied in the range-finding test of the test item. Thirty daphnids in maximum solubility concentration were divided into six groups and exposed for 48 hours.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

EXPERIMENTAL ANIMALS
Species and strain: Daphnia magna laboratory strain
Sex: Female
Source: 100 female D. magna were issued from the National Institute of Pharmacy and were cultured and maintained further in the Test Site’s laboratory.
Justification of species: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 30 animals at each group (concentration and untreated control) divided into 6 replicates (5 animals/1 parallel)
Age of animals: Less than 24 hours old at the beginning of the test. The first breeding of the previous generation was not used.
Acclimatization: To avoid the necessity of adaptation prior to the test, the environmental conditions used in the experiment were similar to the culture conditions (water temperature, pH and light period).
Holding water: Same water was used as test water (see 4.2.3.).
Food and Feeding: The Daphnia group was fed with concentrated algal suspension of Pseudokirchneriella subcapitata. Test animals were not fed during the exposure.
Breeding conditions: Daphnids were cultured in 5 L beakers in an incubator operated for this purpose, with a 12 hours dark-light cycle and temperature control (22 ± 1 °C). The system was located in the Test Site. During the maintenance of the stock, the phenological appearance of the animals were constantly monitored and only healthy animals were kept in the culture.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Tap water from drinking water supply system aerated at least for 24 hours

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

The immobility or mortality of the daphnids was determined by visual observation after 24 and 48 hours. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. Also, other visible abnormalities in appearance or behaviour were recorded.

Test conditions

Test temperature:

22 ± 1 °C

pH:

The pH remained stable during the test in the validity range. For the limit test:
7.35 - 7.91 at the beginning of the test (0 h)
7.90 - 7.95 at the end of the test (48 h)

Dissolved oxygen:

The dissolved oxygen content remained stable during the test in the validity range. For the limit test:
7.89 mg/L - 8.10 mg/L at the beginning of the test (0 h)
7.95 mg/L - 8.10 mg/L at the end of the test (48 h)

Nominal and measured concentrations:

Considering the moderate solubility of the test item, oversaturated solutions were prepared using 400 mg/L test item (nominal concentration). The practical mean concentration of the saturated solution was 328.4 mg/L (average at the beginning of the test)

Details on test conditions:

Experimental design
Female daphnids containing eggs were separated and neonates were collected 24 h prior the test. 100 mL beakers were used during the test. Test vessels were numbered and the following identifiers were indicated: study phase code, test material, concentration, iteration and date. Test solutions were prepared using an “A” signed graduated glass cylinder. The solutions were homogenized in a beaker and 50-50 mLs were poured in the vessels.
Test conditions
Light: 12 hours light and 12 hours dark periods
Water: aerated tap water
Temperature: 22 °C ± 1 the temperature was daily monitored in the climate chamber.
Oxygen concentration: dissolved oxygen was measured at the beginning and end of the test in the control(s) and in the highest test substance concentration
Feeding: no feeding was required during the test
Study phase description: The tests were performed in a static system.
Treatment description: Before the test, the test vessels were labelled and filled with 50 mL of the test solutions. Five (limit test) or ten (preliminary test) 24 h old daphnids were placed in the test solutions.
In the preliminary range-finding test, test substances were assessed in triplicates in 5 concentrations. A control group in six replicates was placed in aerated tap water. The system remained static during the 48 h exposure time. The assessment was designed based on the OECD guide.
In the Limit test, test substance was assessed in one concentration – equivalent to the highest applied concentration in the range-finding test – in six replicates. A control group in six replicates was placed in aerated tap water. The system remained static during the 48 h exposure time. The assessment was designed based on the OECD guide.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 322.2 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 322.2 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

322.2 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

mobility

Details on results:

RESULTS OF THE LIMIT TEST
No immobilised daphnids, or any other adverse effects (including abnormal behaviour) were observable during the test at the control and maximum concentration at any exposure time.

Results with reference substance (positive control):

Reference Control
For the evaluation of the quality of the Daphnia clones and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Latest test with potassium-dichromate (K2Cr2O7) reference substance was performed last at 04.02.2021. The result of the test originated from DAQ2104 GLP test showed that 24h LC50 = 1 mg/L.

Any other information on results incl. tables

RESULTS OF THE RANGE-FINDING TEST

No immobilised daphnids, or any other adverse effects (including abnormal behaviour) was observable during the test at any concentrations and at any exposure time.

Summary Results are presented in Table 1.

Table 1.: Number and percentage of immobilised daphnids

Test item concentration (mg/L)	Number of treated animals	number of Immobilised animals after 24hours	percent (%) of immobilised animals after 24 hours	number of Immobilised animals after 48 hours	percent (%) of immobilised animals after 48 hours
TO	30	0	0	0	0
TO/10	30	0	0	0	0
TO/100	30	0	0	0	0
TO/1000	30	0	0	0	0
TO/10.0000	30	0	0	0	0
Control	60	0	0	0	0

TO = Stock solution (maximum solubility of the test item)

The pH and dissolved oxygen content remained stable during the test in the validity range. Results are presented in Table 2.

Table 2.: pH and dissolved oxygen concentration content

	Test item concentration(mg/L)	ReplicateA	ReplicateB	ReplicateC	ReplicateD	ReplicateE	ReplicateF
Test Start (0h)	TO	pH = 7.14 DO=7.8mg/L	pH = 7.86 DO=7.9mg/L	pH = 7.53 DO=7.7mg/L	-	-	-
	Control	pH = 7.46 DO=8.31mg/L	pH = 7.55 DO=8.41mg/L	pH = 7.58 DO=8.4mg/L	pH = 7.62 DO=8.4mg/L	pH = 7.63 DO=8.4mg/L	pH = 7.65 DO=8.4mg/L
Test End (48h)	TO	pH = 7.65 DO=8.1mg/L	pH = 7.65 DO=8.02mg/L	pH = 7.65 DO=8.04mg/L	-	-	-
	Control	pH = 7.40 DO=8.2mg/L	pH = 7.42 DO=8.2mg/L	pH = 7.48 DO=8.2mg/L	pH = 7.53 DO=8.21mg/L	pH = 7.55 DO=8.24mg/L	pH = 7.56 DO =8.22mg/L

TO= Stock solution (maximum solubility of the test item)

DO = Dissolved Oxygen

As the preliminary range-finding test showed no toxic effect of the test item in D. magna, a limit test was performed. 

RESULTS OF THE LIMIT TEST

Concentration of the test item was analytically determined at the start and at the end of the test. A geometric mean concentration of 322.2 mg/L was calculated based on the measurements.

No immobilised daphnids, or any other adverse effects (including abnormal behaviour) were observable during the test at the control and maximum concentration at any exposure time. Results are presented in Table 3.

 

Table3.:Number and percentage of immobilised daphnids

Mean testitemconcentration(mg/L)	Number of treated animals	number of Immobilised animals after 24 hours	percent (%) of Immobilised animals after 24 hours	number of Immobilised animals after 48 hours	percent (%) of Immobilised animals after 48 hours
322.5	30	0	0	0	0
Control	30	0	0	0	0

The pH and dissolved oxygen content remained stable during the test in the validity range. Results are presented in Table 4.

Table 4.: pH and dissolved oxygen concentration content

	Mean test item concentration (mg/L)	Replicate A	Replicate B	Replicate C	Replicate D	Replicate E	Replicate F
Test Start (0 h)	322.5	pH = 7.91 DO = 8.0 mg/L	pH = 7.87 DO = 7.9 mg/L	pH=7.89 DO =7.89mg/L	pH=7.86 DO =8.00mg/L	pH=7.35 DO =8.00mg/L	pH=7.36 DO =8.10mg/L
	Control	pH=7.33 DO =8.10mg/L	pH=7.35 DO =8.00mg/L	pH = 7.34 DO = 8.1 mg/L	pH=7.36 DO =8.00mg/L	pH = 7.35 DO = 7.9 mg/L	pH=7.36 DO =8.05mg/L
Test End (48 h)	322.5	pH=7.90 DO =7.95mg/L	pH=7.91 DO =7.96mg/L	pH=7.90 DO =8.00mg/L	pH=7.91 DO =8.00mg/L	pH=7.95 DO =8.00mg/L	pH=7.93 DO =8.10mg/L
	Control	pH=7.80 DO =7.91mg/L	pH=7.85 DO =7.90mg/L	pH=7.90 DO =8.00mg/L	pH=7.89 DO =7.92mg/L	pH=7.86 DO =7.90mg/L	pH=7.90 DO =7.90mg/L

DO: Dissolved Oxygen

 

 

Overall, the test item showed no adverse effect in the test system.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this 48-hour acute toxicity test with Daphnia magna the 48 h EC50 and EC100 values were determined to be higher than 322.2 mg/L, and the 48 h NOEC was determined as 322.2 mg/L. The results are based on the measured, calculated test item concentrations.

Executive summary:

The aim of the study was to evaluate the acute toxicity of the test material using the Daphnia sp., acute immobilisation test, conducted on Daphnia magna, under GLP circumstances. 

The study was conducted in accordance with OECD Guideline for testing of chemicals on Daphnia sp., Acute Immobilisation Test (adopted by the council on 13th April 2004). A limit test was performed. The test animals were exposed to aqueous test media containing the test item for 48 hours at only one technical concentration (average 328.4 mg/L at the beginning of the test) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least this test concentration. Considering the information about the stability of the test item, a static test was performed. Significant toxic effect was not observed during the preliminary range-finding test, therefore only one test concentration and one control group was tested in a limit test. Due to the moderate solubility of the test item, the mean of the measured concentrations was calculated in order to determine exposure concentration. Samples were taken and analysed from the test concentration and the control at the start, and at the end of the experiment. The quantification of the test item was performed by the analytical laboratory of the test facility.

In this 48-hour acute toxicity test with Daphnia magna the 48 h EC50 and EC100 values were determined to be higher than 322.2 mg/L, and the 48 h NOEC was determined as 322.2 mg/L. The results are based on the measured, calculated test item concentrations. There was no immobilization in the daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Due to the moderate solubility of the test item, a geometric mean concentration was calculated based on the values measured at the beginning and at the end of the experimental phase. Accordingly, the 48-h NOEC was determined as 322.2 mg/L. The 48-h EC50 and EC100 values were determined to be higher than 322.2 mg/L. All biological results are related to the measured, calculated test item concentration.