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

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:

Start of experiment: 16 February 2021- End of experiment:19 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 201 (Adopted: 23 March 2006)

Deviations:

no

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), in 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 alga 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 alga 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: 50 μL acetonitrile and 900 μL diluent were added to
50 μL sample (20 fold dilution); dilutions were made in 5 replicates
Samples at the END of the alga 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. Test flasks 1-6
contained alga, while the seventh flask contained test item solution without alga.
Approximately 2 mL of the solutions containing alga were filtered through a 0.22 μm syringe
filter (cellulose acetate) before further dilution. Dilution of the samples:
- control 1 solution: 1:1 dilution was prepared with acetonitrile in duplicates
- test item stock solution 1-6 containing alga: 50 μL acetonitrile and 900 μL diluent
were added to 50 μL filtered sample (20 fold dilution); dilutions were made in
duplicates

Test solutions

Vehicle:

yes

Remarks:

Test item dissolution in ultrapure water using sonication or incubation

Details on test solutions:

Preparation of Testing Solutions
Due to the moderate solubility of the test item, oversaturated solutions were prepared in Z8 media using 80 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 inoculation with the algae (start of the experiment).
Preliminary Range-Finding Test
In order to select appropriate test concentrations for use in the definitive test, a preliminary rangefinding test was conducted to determine the approximate toxicity of the test item. The examined concentrations were 46.56 mg/L.
No significant toxic effects were not observed during the preliminary range-finding test, therefore only one test concentration (nominal 80 mg/L, saturated solution) 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 performed to demonstrate that the test item has no toxic effect on the algae up to its limit of solubility in the test water. Thus, a saturated solution of the test item was tested. Additionally, a control was run in parallel (test medium without addition of the test item).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM:
- Species, strain: Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus), 61.81 SAG
Justification of species selection: The species of Desmodesmus subspicatus used, being a fast growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.

- Source (laboratory, culture collection): The algae were cultivated at the test site’s laboratories under standardized conditions according to the test guidelines.

Maintaining of the stocks
Algae strains are cultured in 250 mL Erlenmeyer flasks in a Memmert 110 (no. 1.) incubator operated for this purpose, with continuous illumination (7000-8000 LUX), stirring and temperature control (22±1°C). The system is located in the basement floor of the GLP Testing Site in the Algae and Daphnia Laboratory and Observation Room. Algae strains are cultured in triplicate with a phase shift in order to have accessible cultures in log-phase of growth stage any time. The cultures are refreshed every 10-12 days by inoculating the culture medium using stock cultures maintained on 1% agar medium, with a final concentration of 300.000-400.000 cells/mL. During the maintenance of the stock, the overall condition of the cultures and productional stages are continuously monitored.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

The vessels were held for the duration of the study in a climate chamber, the test temperature was 22 ± 1 °C. The water temperature was determined at least daily in one vessel filled with water only and continuously with a min/max thermometer in the climate chamber

pH:

The pH was measured in the control(s) and in the used test concentration in each test vessel at the start and at the end of the experiment. The pH varied by not more than 1.5 units in any test.
The pH in the vessels with the test substance at the beginning of the test varied from 7.20 to 7.22 and after 72h it varied from 7.65 to 7.70.

Nominal and measured concentrations:

the nominal concentration of the test solution was 80 mg/L, however, the maximal solubility in the media used in the experiment is lower.
Based on the analytical measurements at the beginning and at the end of the experimental phase, the geometric mean concentration of the test item in the test solutions was 50.8 mg/L.

Details on test conditions:

Experimental design
The growth inhibition test was run in a static system layout.
Volumes of 100 mL algal suspension per replicate, in 250 mL Erlenmeyer flasks, were continuously shaken by a laboratory orbital shaker. The flasks were covered with airpermeable stoppers. Ensuring the aeration and mixing was carried out by aeration pumps. The Erlenmeyer flasks individually numbered and the following identifiers were indicated: study phase code, test material, concentration, iteration and date. Test and control solutions were inoculated with algae in log growth phase. The initial biomass in the test cultures was set to 5 x 10^3 - 10^4 cells/mL. The initial biomass of algae was set in the test solutions by absorbance measurement (750 nm) using a spectrophotometer by adding stock algae solution. Absorbance measurement was performed parallel to algae cell counting in Bürker chamber. The obtained cell concentration values for absorbance and chamber counting were used as calibration curve points. Growth rate was measured during the exposure time (72 h).
Optical density was measured in all replicates and treatment groups at the start of the tests, after 24, 48, and 72 h exposure. Also, other visible abnormalities in appearance of the algae cells were recorded.
In the range-finding test, the test was run in triplicates in 5 concentrations plus controls in six replicates. In the limit test, the test was run in six replicates of maximum water solubility concentration and six replicates of control.

Illumination: fluorescent lamps were used with continuous lighting with light intensity of 7’000-8’000 lux at the liquid surface.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50.8 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50.8 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 50.8 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

> 50.8 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

yield

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50.8 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50.8 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

yield

Results with reference substance (positive control):

Reference Control
For the evaluation of the quality of the algae and validation of 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 and showed that EC50 = 0.9 mg/L.

Any other information on results incl. tables

RESULTS OF THE RANGE-FINDINGTEST

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

RESULTS OF THE LIMITTEST

All biological results have been related to the geometric mean of the measured test item concentrations during the experiment.

The test item had no statistically significant inhibitory effect on the growth based on the average specific growth rate and yield of Desmodesmus subspicatus (Table 1 and 2).

 

Table 1.:Determination of grow rates and inhibition

Nominal concentration (mg/L)		N° ofcells (x1000) 0 h	N° ofcells (x1000) 24h	N° ofcells (x1000) 48h	N° ofcells (x1000) 72h	u(0-24)	u(0-48)	u(0-72)	% Inhibition
		5	17.2	62.5	187.5	0.0516	0.0526	0.0503	0.0
		5	16.7	75.0	162.5	0.0502	0.0564	0.0484
		5	16.7	75.0	162.5	0.0502	0.0564	0.0484
Control		5	18.2	75.0	162.5	0.0539	0.0564	0.0484
		5	17.3	75.0	175.0	0.0516	0.0564	0.0494
		5	17.0	75.0	187.5	0.0509	0.0564	0.0503
	Mean	5	17.2	72.9	172.9	0.0514	0.0558	0.0492
	SD	0.0	0.6	5.1	12.3	0.0014	0.0016	0.0010
		5	16.7	137.5	175.0	0.0502	0.0690	0.0494	1.4
		5	17.0	100.0	162.5	0.0509	0.0624	0.0484
		5	17.3	75.0	175.0	0.0516	0.0564	0.0494
50.8		5	17.9	62.5	162.5	0.0531	0.0526	0.0484
		5	17.6	62.5	150.0	0.0524	0.0526	0.0472
		5	16.4	100.0	162.5	0.0495	0.0624	0.0484
	Mean	5	17.1	89.6	164.6	0.0513	0.0593	0.0485
	SD	0.0	0.6	29.0	9.4	0.0014	0.0065	0.0008

 

Table 2.:Determination of yield and inhibition

Nominalconcentration (mg/L)		No of cells (x1000) 0h	No of cells (x1000) 72h	Yield	%Inhibition
Control		5	187.5	182.5	0.0
		5	162.5	157.5
		5	162.5	157.5
		5	162.5	157.5
		5	175.0	170.0
		5	187.5	182.5
	Mean	5	172.9	167.9
	SD	0.0	12.3	12.3
50.8		5	175.0	170.0	5.0
		5	162.5	157.5
		5	175.0	170.0
		5	162.5	157.5
		5	150.0	145.0
		5	162.5	157.5
	Mean	5	164.6	159.6
	SD	0.0	9.4	9.4

Summary of Biological Endpoints

Parameter	Growth rate (r) [mg/L]	Yield (y) [mg/L]
Calculation based on the geometric mean of the measured concentrations
72-hEC50	>50.8 (nominalconcentration: 80mg/L)	>50.8 (nominalconcentration: 80mg/L)
72-hEC100	>50.8	>50.8
72-hNOEC	50.8	50.8

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item showed no adverse effect in the test system. In the 72-h algal growth inhibition test on Desmodesmus subspicatus, the 72-h EC50 value, based on growth rate was determined to be higher than 50.8 mg/L and based on yield was calculated to be higher than 50.8 mg/L. The NOEC related to growth rates was determined to be 50.8 mg/L and related to yield it was determined to be 50.8 mg/L. The results are based on the geometric mean of the measured concentrations.

Executive summary:

The purpose of this study was to determine the effect of the test item on the growth of a unicellular green algal species Desmodesmus subspicatus. Exponentially growing cultures of Desmodesmus subspicatus were exposed to the test item over several generations under defined conditions. As no symptoms or inhibition of grow rate were observed during the preliminary study, a limit test was performed. The test design included six replicates of the test concentration and six replicates for the untreated controls.

Due to the moderate solubility of the test item, oversaturated solutions were prepared in the algae media with 80 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. Therefore, the nominal concentration of the test solution was 80 mg/L, however, the maximal solubility in the media used in the experiment is lower. Based on the analytical measurements at the beginning and at the end of the experimental phase, the geometric mean concentration of the test item in the test solutions was 50.8 mg/L. All biological results are based on this geometric mean of the measured concentrations.

The initial biomass in the test cultures was set to 5 x 10^3 cells/mL. Glass flasks with total capacity of ~250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The test vessels were covered with air-permeable stoppers.