Insulin DesB30

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)

Remarks:

(Human Insulin Methyl Ester S5)

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 DesB30 to fish is assumed to follow the same pattern as that of the source substance because of the high degree of structural similarity:
• Insulin DesB30 and the source substance belong to the same chemical categories: human insulins, polypeptides and proteins
• Insulin DesB30 and the source substancs 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:

LOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.38 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.:

> 7.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

not applicable

Conclusions:

The aquatic toxicity of Insulin DesB30 is assumed to be similar to the source substance (S5) because of the high degree of structural similarity.
•       Insulin DesB30 and the source substance belong to the same chemical categories: human insulins, polypeptides and proteins
•       Insulin DesB30 and the source substancs are composed of polypeptide containing amino acids

Based on the results obtained during the study with Insulin Human Methyl Ester (S5) an NOEC, EC10 and EC50 (48h) of <0.25, 0.33 and > 7.23 mg/L (geom. mean) were determined.

Executive summary:

The aquatic toxicity of Insulin DesB30 is assumed to be similar to the source substance (S5) because of the high degree of structural similarity.

•       Insulin DesB30 and the source substance belong to the same chemical categories: human insulins, polypeptides and proteins

•       Insulin DesB30 and the source substancs are composed of polypeptide containing amino acids

Based on the endpoint growth rate a LOEC and a NOEC of 0.31 mg/L and <0.25 mg/L respectively and an EC10, EC20 and EC50 (72h) of 0.33, 0.38 and > 7.23 mg/L (geom. mean concentration) were determined during the study.

Insulin DesB30 is predicted to have a toxicity to algae with NOEC, EC10 and EC50 (48h) of <0.25, 0.33 and > 7.23 mg/L

based on the results from the study with Insulin Human Methyl Ester.

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: Hi-OMe (Human Insulin Methylester)

Chemical name: Zinc crystals of HI-OMe
CAS No: Not applicable
Molecular formula: C258H385N65O77S6
Purity: 95-98%
Impurities: Derivatives of HI-OMe, desB30-Insulin, Insulin Human
Percentage of (significant) main impurities:
Typical Concentration: <3.0 % w/w
Concentration Range: 2.0 – 5.0 % w/w
Form: White powder
Water solubility: Dependent of pH
Log Pow: Not possible to perform

Batch Number: EM0Z31503
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 pre-pared 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); 0.2; 0.5; 1.0; 2.0; 10 and 20 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.

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 will be used in the study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not included

Test temperature:

23 ± 2.0°C

pH:

8.1 ± 0.2

Nominal and measured concentrations:

The following concentrations were applied: 0 (control); 0.2; 0.5; 1.0; 2.0; 5.0; 10 and 20 mg/L
Measured concentrations:

Details on test conditions:

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

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.38 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.:

> 7.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Results based on nominal concentrations
NOEC: 1.0 mg/L
LOEC: 2.0 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 the endpoint growth rate a LOEC and a NOEC of <0.25 mg/L and 0.31 mg/L respectively and an EC10, EC20 and EC50 (72h) of 0.33, 0.38 and > 7.23 mg/L (geom. mean concentration) were determined during the study.

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 concentrations were applied: 0 (control); 0.2; 0.5; 1.0; 2.0; 5.0; 10 and 20 mg/L.

Concentrations were not within 20% of the nomial concentration throughout the study. And endpoints are thus reported as geometric mean concentrations (geo. mean conc.)

Based on the endpoint growth rate a LOEC and a NOEC of 0.31 mg/L and <0.25 mg/L respectively and an EC10, EC20 and EC50 (72h) of 0.33, 0.38 and > 7.23 mg/L (geom. mean concentration) were determined during the study.

Description of key information

The ecotoxicity of Insulin DesB30 is assumed to be simmilar to the source substance (S5) because of the high degree of structural similarity.

Based on the results obtained during the study with Insulin Human Methyl Ester (S5) a LOEC and a NOEC of 0.31 mg/L and <0.25 mg/L respectively and an EC10, EC20 and EC50 (72h) of 0.33, 0.38 and > 7.23 mg/L (geom. mean concentration) are reported.

Insulin DesB30 is predicted to have a toxicity towards algae of EC10 = 0.33 and EC50 of > 7.23 mg/L (geometric mean concentration).

Key value for chemical safety assessment

EC50 for freshwater algae:

7.23 mg/L

EC10 or NOEC for freshwater algae:

0.33 mg/L