Insulin Aspart Ethyl Ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read Across please refer to section "justification for type of information"

Justification for type of information:

The aquatic toxicity of Insulin Aspart Ethyl Ester to algae is assumed to follow the same pattern as that of the source substance because of the high degree of structural similarity:
• Insulin Aspart Ethyl Ester and the source substance belong to the same chemical categories: human insulins, polypeptides and proteins
• Insulin Aspart Ethyl Ester and the source substance are composed of polypeptide containing amino acids

Please refer to Environmental Assessment Reports (Section 13)

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 258 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 258 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

not applicable

Conclusions:

The aquatic toxicity of Insulin Aspart Ethyl Ester is assumed to be similar to the source substance (S4) because of the high degree of structural similarity:
•       Insulin Aspart Ethyl Ester and the source substance belong to the same chemical categories: human insulins, polypeptides and proteins
•       Insulin Aspart Ethyl Ester and the source substance are composed of polypeptide containing amino acids.

Insulin Aspart Ethyl Ester is predicted to have a toxicity to algae with EC10 and EC50 of > 258 mg/L based on the results from the study with Insulin Aspart Precursor. No effects to algae were observed during the study with Insulin Aspart Precursor (S4) and EC10 and EC50 are therefore reported as > 300 mg/L (> 258 mg/L geometric mean concentration).

Executive summary:

The aquatic toxicity of Insulin Aspart Ethyl Ester is assumed to be similar to the source substance (S4) because of the high degree of structural similarity:

•       Insulin Aspart Ethyl Ester and the source substance belong to the same chemical categories: human insulins, polypeptides and proteins

•       Insulin Aspart Ethyl Ester and the source substance are composed of polypeptide containing amino acids.

Insulin Aspart Ethyl Ester is predicted to have a toxicity to algae with EC10 and EC50 of > 258 mg/L based on the results from the study with Insulin Aspart Precursor. No effects to algae were observed during the study with Insulin Aspart Precursor (S4) and EC10 and EC50 are therefore reported as > 300 mg/L (> 258 mg/L geometric mean concentration).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006.03.23

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

2012.02.15

GLP compliance:

yes

Specific details on test material used for the study:

The test item is an intermediate. Based on information from the sponsor, the test item is:
The test item is: Insulin Aspart (IA) Precursor (NN2000 precursor)
Chemical name: Iso precipitate of IA precursor
CAS No: Not applicable
Molecular formula: C302H446N76O91S7
Purity: 90-99%
Impurities: Derivatives of IA precursor
Percentage of (significant) main impurities:
O-glycosylated derivatives, desamido derivatives and ox-idated derivatives
Typical concentration: 4.0 % w/w
Concentration Range: 1.0 – 10.0 % w/w
Form: White powder
Water solubility: Dependent of pH
Log Pow: Not possible to perform
Batch Number: K2, FHIAZG135 (undissolved substance)
Manufacturing date: 27.11.2016
Stability: In aqueous solution, stable up to 96 hours at 4°C
Origin: Novo Nordisk A/S

Analytical monitoring:

yes

Details on sampling:

One flask of the control and one flask of each test concentration without algae was used to collect samples for chemical analysis.
Duplicate samples of approx. 5 mL from the specially prepared test flasks were collected in 20-mL plastic vials at the initiation of the test and at each sampling time (24h; 48h and 72h). Samples for chemical analysis were anonymized and only test item and concentration range was indicated on the samples. The samples taken were stored at -20 ± 2.0°C and sent frozen to the analytical laboratory.

Samples relevant for the calculation of the No Observed Effect Concentration (NOEC) and the ECx concentrations were analysed.

Although duplicate samples were taken of each of the test concentrations, only one of these duplicate samples was analysed, if relevant. The other sample served as a spare sample and was discharged after sponsor’s acknowledgement of the report.

Vehicle:

no

Details on test solutions:

The test item was tested at the following nominal concentrations: 0 (control); 5; 10; 20; 40; 75; 150 and 300 mg/L
The reference compound potassium dichromate (K2Cr2O7) was tested in the following concentrations: 0 (control); 0.1; 0.2; 0.4; 0.7; 1.0; 1.4 and 2.0 mg/L.

The test was run in 250-mL conical glass flasks capped with glass beakers. Each glass flask contained approx. 100 mL of test solution.

The test included three replicates of each concentration of the test item. Six control replicates was used. One flask of the control and each test concentration without algae served as blank controls during the test when measuring the fluorescence. Furthermore, one flask of the control and one flask of each test concentration without algae was used to collect samples for chemical analysis.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata (NIVA-CHL 1) is cultured at DHI. Algae from this culture was used in the study.
At the start of the test, the cell density was adjusted to approx. 6,000-10,000 cells/mL by use of a Beckman Coulter Multisizer. At the initiation of the test and at 24; 48 and 72 ± 2 hours, the in-vivo fluorescence was measured on sub-samples from each test flask by use of a Turner fluorometer.
The test flasks were stored under continuous shaking at approx. 100 rpm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not included

Test temperature:

22.3 ± 0.1°C in a climate room

pH:

pH at start: 7.9; pH at termination 7.9-9.4.

Nominal and measured concentrations:

Nominal concentrations: 0 (control); 5; 10; 20; 40; 75; 150 and 300 mg/L
Measured concentrations (Geom. mean): 0; 2.6; 6.6; 15.3; 32.3; 64.7; 129.6 and 257.4 mg/L

Details on test conditions:

illumination from a panel of fluorescent light with an intensity of 60-120 µmol/m2 × s

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The corresponding geometric mean concentration was determined to 258 mg/L

Results with reference substance (positive control):

Potassium dichromate (K2Cr2O7)
NOEC = 0.1 mg/L
LOEC = 0.2 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Based on growth rate, a NOEC of 258 mg/L and a LOEC > 258 mg/L (geom. mean concentration) were determined.
Data did not allow statistical calculation of the limit of the 95% confidence interval. EC10, EC20 and EC50 (72h) are all reported as >300 mg/L (nominal concentration) (>258 mg/L geom. mean concentration)

Executive summary:

A Freshwater Alga and Cyanobacteria, Growth Inhibition Test was conducted according to Guideline no 201. The study was conducted according to GLP.

The following nominal concentrations were applied: 0 (control); 5; 10; 20; 40; 75; 150 and 300 mg/L

Based on growth rate a NOEC of 300 mg/L (258 mg/L geom. mean), a LOEC > 300 mg/L (>258 mg/L geom. mean) and an EC10, EC20 and EC50 (72h) >300 mg/L (>258 mg/L geom. mean) were determined during the study.

Description of key information

The ecotoxicity of Insulin Aspart Ethyl Ester is assumed to be simmilar to the source substance (S4) because of the high degree of structural similarity.

No mortality towards fish was observed during the study with Insulin Aspart Precursor (S4) and EC10 and EC50 are therefore reported as > 300 mg/L (> 257.4 geometric mean concentration).

Insulin Aspart Ethyl Ester is predicted to have a toxicity towards algae of > 300 mg/L (nominal) (257.4 mg/L active substance).

Key value for chemical safety assessment

EC50 for freshwater algae:

257.4 mg/L

EC10 or NOEC for freshwater algae:

257.4