Insulin Human Methyl Ester

Water solubility

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

water solubility

Data waiving:

other justification

Justification for data waiving:

other:

Key result

Water solubility:

> 10 g/L

Temp.:

20 °C

pH:

> 6.5

Remarks on result:

other: Insoluble at pH 4.5-6.5. Outside this pH range solublity is larger than 10 g/L

Key result

Water solubility:

> 10 g/L

Temp.:

20 °C

pH:

< 4.5

Remarks on result:

other: Insoluble at pH 4.5-6.5. Outside this pH range solublity is larger than 10 g/L

Conclusions:

Insulin is insoluble at pH 4.5-6.5. Outside this pH range solublity is larger than 10 g/L. The solubility is was estimated to be >10g/L, no experimental study was performed.

Executive summary:

Insulin is insoluble at pH 4.5-6.5. Outside this pH range solublity is larger than 10 g/L. The solubility is was estimated to be >10g/L, no experimental study was performed.

Reference 2

Endpoint:

water solubility

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Water solubility:

> 10 g/L

Remarks on result:

not measured/tested

Remarks:

Based on read across to S2 and S3

Conclusions:

Data on target substance not available. Thus, read-across has been applied using data from source substances (S1, S2 and S3).

As can be seen from the molecular structure (see attached document in section 13) the target substance is a small protein consisting of 51 amino acids have at least 48 amino acids in common with the source substances (consisting of 50-53 amino acids) and having very identical amino acid sequences. Based on these structural similarities very similar physical chemical properties would be expected. This is further documented by the results from physical chemical guideline testing for S2 and S3 where the test results found for melting point, boiling point, flammability, self-ignition, and explosion were identical.

The water solubility of MI3 and X14DesB30 (solution) were determined by the shake flask method. The procedure conformed to EC Directive 92/69/EEC Method A6, and OECD Guideline 105 (1995). The solubility of MI3 at 20oC in double distilled water was determined to be 393.1 mg/L±44.0 mg/L with a resultant pH of nominally 7.9. The solubility of X14DesB30 at 20oC in double distilled water was determined to be 43 g/L at pH 3.6. However, Human insulin was insoluble in the pH range of 4.5 – 6.5, but outside this it had a solubility >10g/L. Therefore, based on read-across it is concluded that the solubility of the target substance (Insulin human methyl ester) is very pH dependant with a solubility of about 43 g/L at pH 3.6 and 393 mg/L at pH 7.9. In the interval of pH 4.5-6.5 the substance can be considered as insoluble.

Executive summary:

Data on target substance not available. Thus, read-across has been applied using data from source substances (S1, S2 and S3).

As can be seen from the molecular structure (see attached document in section 13) the target substance is a small protein consisting of 51 amino acids have at least 48 amino acids in common with the source substances (consisting of 50-53 amino acids) and having very identical amino acid sequences. Based on these structural similarities very similar physical chemical properties would be expected. This is further documented by the results from physical chemical guideline testing for S2 and S3 where the test results found for melting point, boiling point, flammability, self-ignition, and explosion were identical.

The water solubility of MI3 and X14DesB30 (solution) were determined by the shake flask method. The procedure conformed to EC Directive 92/69/EEC Method A6, and OECD Guideline 105 (1995). The solubility of MI3 at 20^oC in double distilled water was determined to be 393.1 mg/L±44.0 mg/L with a resultant pH of nominally 7.9. The solubility of X14DesB30 at 20^oC in double distilled water was determined to be 43 g/L at pH 3.6. However, Human insulin is insoluble in the pH range of 4.5 – 6.5, but outside this it has a solubility >10g/L.  Therefore, based on read-across it is concluded that the solubility of the target substance (Insulin human methyl ester ) is very pH dependant with a solubility of about 43 g/L at pH 3.6 and 393 mg/L at pH 7.9. In the interval of pH 4.5-6.5 the substance can be considered as insoluble.

Reference 3

Endpoint:

water solubility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.03.16 – 2000.09.18

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

Deviations:

yes

Remarks:

Deviation in the incubation temperature measured to be 18.6 oC instead of 19.5 to 20.5 oC as specified by the guideline. This deviation is considered to have little impact on the test results.

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Deviations:

yes

Remarks:

Deviation in the incubation temperature measured to be 18.6 oC instead of 19.5 to 20.5 oC as specified by the guideline. This deviation is considered to have little impact on the test results.

GLP compliance:

yes (incl. QA statement)

Type of method:

flask method

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (MI3 solution)
- Appearance: brown paste
- Source and lot/batch No.of test material: QC202426
- Expiration date of the lot/batch: June 2004
- Purity test date: 2003-06-18
- Purity: 84% w/w (stated ny Sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Frozen below - 10°C

Key result

Water solubility:

ca. 393 mg/L

Conc. based on:

test mat. (dissolved fraction)

Incubation duration:

ca. 24 - ca. 72 h

Temp.:

30 °C

pH:

ca. 7.8 - ca. 8.06

Remarks on result:

other: Standard deviation of +/- 44 mg/mL

Calibration data is presented in the table below; the calibartion line was established linearly with 1/x weighting. 

Sequence reference	WSOL2
Slope	46441.6
Intercept	-10980.08
Correlation	0.99983
Minimum calibrant conc (µg/ml)	0.5
Maximum calibrant conc (µg/ml)	50

Analytical data is shown in the table below. The incubation bath was constant at a temperature of 18.6^oC over the test period.

Test ref	Time at 30^oC (h)	pH	Dilution factor	Peak area	MI3 conc (mg/L)
1	72	7.80	100	139980.1	325.05
2	72	7.94	100	204180.4	463.29
3	48	7.97	100	174576.7	399.55
4	48	7.97	100	168503.7	386.47
5	24	8.06	100	169386.6	388.37
6	24	8.02	100	172798.3	395.72

  

Conclusions:

The water solubility of MI3 (solution) was determined by the shake flask method. The procedure conformed to EC Directive 92/69/EEC Method A6, and OECD Guideline 105 (1995). The solubility of MI3 at 20 oC in double distilled water was determined to be 393.1 mg/L ± 44.0 mg/L with a resultant pH of nominally 7.9.

Executive summary:

The water solubility of MI3 (solution) was determined by the shake flask method. The procedure conformed to EC Directive 92/69/EEC Method A6, and OECD Guideline 105 (1995). The solubility of MI3 at 20^oC in double distilled water was determined to be 393.1 mg/L±44.0 mg/L with a resultant pH of nominally 7.9. This is equivalent to a CV of 11.2%, which is within the recommended limit (±15 %), and therefore considered acceptable especially when keeping in mind that the incubation bath temperature of 18.6^oC, was outside the specified temperature of 20^oC±0.5^oC.

Reference 4

Endpoint:

water solubility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.03.10 - 2000.09.21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

other: miscibility test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (X14desB30)
- Appearance: Brown solid when frozen, brown paste when thawed.
- Source and lot/batch No.of test material: X14ISO201014
- Expiration date of the lot/batch: NA
- Purity test date: 2000-02-18
- Purity: 155 mg/g Active Ingredient (stated by sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stored < - 10 degree celcius.

Key result

Water solubility:

ca. 43 - ca. 56.17 g/L

Conc. based on:

test mat. (total fraction)

Temp.:

20 °C

pH:

ca. 3.6

Conclusions:

Determination of the water solubility of X14desB30 was performed using a miscibility test in accordance to OECD guideline 105 (1995). The solubility of X14DesB30 at 20oC in double distilled water was evaluated to be 43 g/L at pH 3.6.

Executive summary:

Determination of the water solubility of X14desB30 was performed in accordance to OECD guideline 105 (1995) and EC Directive 92/69/EEC Methods A6. The test substance was inhomogeneous in suspension in water, hence the flask method and column elution method was not applicable and a miscibility test was performed. The solubility of X14DesB30 at 20^oC in double distilled water was evaluated to be 43 g/L at pH 3.6.

Description of key information

Data on target substance not available. Thus, read-across has been applied using data from source substances (S1, S2 and S3).

As can be seen from the molecular structure (see attached document in section 13) the target substance is a small protein consisting of 51 amino acids have at least 48 amino acids in common with the source substances (consisting of 50-53 amino acids) and having very identical amino acid sequences. Based on these structural similarities very similar physical chemical properties would be expected. This is further documented by the results from physical chemical guideline testing for S2 and S3 where the test results found for melting point, boiling point, flammability, self-ignition, and explosion were identical.

Key value for chemical safety assessment

Water solubility:

393 mg/L

at the temperature of:

20 °C

Additional information

The water solubility of MI3 and X14DesB30 (solution) were determined by the shake flask method. The procedure conformed to EC Directive 92/69/EEC Method A6, and OECD Guideline 105 (1995). The solubility of MI3 at 20^oC in double distilled water was determined to be 393.1 mg/L±44.0 mg/L with a resultant pH of nominally 7.9. The solubility of X14DesB30 at 20^oC in double distilled water was determined to be 43 g/L at pH 3.6. However, Human insulin is insoluble in the pH range of 4.5 – 6.5, but outside this it has a solubility >10g/L.  Therefore, based on read-across it is concluded that the solubility of the target substance (Insulin human methyl ester) is very pH dependant with a solubility of about 43 g/L at pH 3.6 and 393 mg/L at pH 7.9. In the interval of pH 4.5-6.5 the substance can be considered as insoluble.