Insulin Aspart Precursor

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The conducted in vitro mutagenicity testing on MI3, X14DesB30 and insulin aspart do not indicate any mutagenic or clastogenic potential of the substances. Due to very close structural similarity to insulin aspart precursor a lack of mutagenic and clastogenic potential in vitro can be concluded for this substance as well.

See read-across template and justification attached in section 13

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EEC Methd B14 specified in the Annex to 92/69/EEC

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990)

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline (1997)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

MI3, solid, purity of 98.4%

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: Histidine dependent

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) prepared from Sprague Dawley rats induced with Arochlor 1254

Test concentrations with justification for top dose:

Two experiments were carried out:

Range finder experiment and mutation experiment 1:
Test concentrations: 1.6, 8, 40,200,1000 and 5000 ug/mL

Mutation experment 2:
Test concentrations: 156.25, 312.5, 625, 1250, 2500 and 5000 ug/mL

Vehicle / solvent:

Due to the proteineous nature of the test substance, sterile water was considered to be the most appropriate solvent for this study.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene and glutaraldehyde

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Range-finder experiment:
S. typhimurium TA100 were exposed in triplicates to the above mentioned concentrations with and without S-9 (4 solvent controls and triplicate positive controls) for 3 days. Plates were investigated for toxicity and where possible revertant colonies were counted.

Mutation experiments:
All five strains were tested in triplicates with and without S-9.

Preincubation period: 1 hour in experiment 2

Evaluation criteria:

The test article was considered to be mutagenic if:
1. the assay was valid
2. Dunnett's test gave a significant response (p ≤ 0.01) and the data ste(s) showed a significant dose correlation
3. the positive responses described above were reproducible

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was chekced by linear regression analysis.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of MI3 at 1.6, 8, 40, 200, 1000 and 5000µg/plate plus solvent and positive controls. Following these treatments and those of the remaining strains in experiment 1, no evidence of toxicity was observed.

In experiment 1, maximum test dose treatments of strain TA1537 (with and without S-9) resulted in several microcolonies in addition to the characteristic larger colonies normally observed with this strain. Streaks of bith types of colony types on to minimal agar (with biotin) plates and on to nutrient agar plates confirmed that only the larger bacterial colonies were true revertants. The plates were therefore scored manually to only count the larger colonies. The presence of microcolonies were not considered to have affected the integrity of this assay and the manual counts were accepted as valid.

Experiment 2 treatments retained 5000 µg/plate as the maximum test done but employed a narrowed dose range in order to investigate those doses of MI3 considered most likely to induce mutagenic response. In addition, treatments with S-9 were modified by the inclusion of a pre-incubation step. In this way it was hoped to increase the range of mutagenic chemicals that would be detected in the assay system. No signs of toxicity was observed in any test strains.

Negative (solvent) controls and positive controls were included for all strains in both experiments. The mean number of revertant colonies on negative control plates all fell within acceptable ranges and were significantly elevated by positive control treatments

Conclusions:

MI3 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under conditions employed in this study. The conditions included treatments at concentrations up to 5000 ug/plate, both in the absence and in the presence of a rat liver metabolic activation system (S-9)

Executive summary:

MI3 was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of salmonella typhimurium, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9) in two separate experiments.

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of MI3 at 1.6, 8, 40, 200, 1000 and 5000µg/plate plus solvent and positive controls. Following these treatments and those of the remaining strains in experiment 1 and 2, no evidence of toxicity was observed.

MI3 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under conditions employed in this study. The conditions included treatments at concentrations up to 5000 ug/plate, both in the absence and in the presence of a rat liver metabolic activation system (S-9)

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EEC Methd B14 specified in the Annex to 92/69/EEC

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990)

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline (1997)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

X14DesB30, solid, purity of 85.1%

Target gene:

S. typhimurium: histidine
E. coli: tryptophane

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: Histidine-requiring

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: tryptophane-requiring

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Additional strain / cell type characteristics:

other: tryptophne-requiring

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) prepared from Sprague Dawley rats induced with Arochlor 1254

Test concentrations with justification for top dose:

Two exeriments were carried out:

Mutation experiment 1:
Test concentrations: 1.6, 8, 40,200,1000 and 5000 ug/mL

Mutation experment 2:
Test concentrations: 156.25, 312.5, 625, 1250, 2500 and 5000 ug/mL

Vehicle / solvent:

Due to the proteineous nature of the test substance, sterile water was considered to be the most appropriate solvent for this study.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

Remarks:

Other positive control substances: 2-aminoanthracene, N-methyl-N-nitro-N-nitrosguanidine and ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
All plating were achieved by the addition of bacteria to 0.9% (w/v) soft agar For the S. typhimurium strains, this agar contained 10 ug/mL histidine HCl and 12.2 ug/mL d-biotin-. For the E. coli strains the agar contained 6.45 ug/mL DL-tryptophan and 310 ug/mL casamino acids.


Mutation experiments:
All five strains were tested in triplicates with and without S-9.

Preincubation period: 1 hour in experiment 2

Evaluation criteria:

The test article was considered to be mutagenic if:
1. the assay was valid
2. Dunnett's test gave a significant response (p ≤ 0.01) and the data ste(s) showed a significant dose correlation
3. the positive responses described above were reproducible

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was chekced by linear regression analysis.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: WP2 uvrA pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Experiment 1 treatments of all the test strains employed final concentrations of X14DesB30 at 1.6, 8, 40, 200, 1000 and 5000 ug/mL both with and witout S-9, plus positive and negative controls. No evidence of toxicity (as would normally be indicated by a thinning of the background bacterial lawn or a reduction in revertant numbers) was observed following any of these treatments. Furthermore, the viable cell counts for thesee treatments also demonstrated no evidence of test article indiced toxicity.

In experiment 2, 5000 ug/ml was retained as the maximum test dose for treatment of all test strains. A narrowed dose range (156.25 -5000 ug/ml) was employed in order to investigate those concentrations of X14DesB30 considered most likely to induce mutation. As in experiment 1, no toxic signs (either evident on the mutation plates or determined by the viable cell counts) were observed in any of the test strains following these experiment 2 treatments.

No X14DesB30 treatments of any of the test strains induced a statistically significant increase in revertant numbers, when the data were analysed at the 1% level using Dunnett's test. This study has provided no evidence of any X14DesB30 mutagenic activity.

Conclusions:

X14DesB30 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535, andTA1537) of salmonella typhimurium, and two tryptophan-requiring strains (WP2 pKM101 and WP2 uvrA pKM101) of Escherichia coli, when tested under the onditions employed in this study. The conditions included treatments at concentrations up to 5000 µg/mL, both in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

ÆNDRES til X14DesB30

X14DesB30 was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535, andTA1537) of salmonella typhimurium, and two tryptophan-requiring strains (WP2 pKM101 and WP2 uvrA pKM101) of Escherichia coli, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9) in two separate experiments.

A 'treat and plate' procedure was used for all treatments in this study as X14DesB30 is known to be a protein (which may cause artefacts through growth stimulation in a standard plate-incorporation test).

Experiment 1 treatments of all the test strains employed final concentrations of X14DesB30 at 1.6, 8, 40, 200, 1000 and 5000µg/mL plus negative (solvent) and positive controls. Following these treatments and those of the remaining strains in experiment 1 and 2, no evidence of toxicity was observed. No evidence of toxicity was observed following any of these treatments.

In experiment 2, 5000 µg/mL was retaied as the maximum test dose for treatments of all test strains. A narrowed dose range (156.6 -5000 µg/L) was employed in order to investigate those concentrations of X14DesB30 considered most likely to induce any mutation. As in Experiment 1, no toxic signs were observed in any of the trains following these Experiment 2 treatments.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Negative (solvent) and positive controls were included for all strains in both experiments. The mean number of revernant colonies on negative control plates were all comparable with acceptable ranges, and were sufficiently elevated by positive control treatments.

No X14DesB30 treatments of any of the test strains induced an increase in revertant numbers that could be considered indicative of test article mutagenic activity.

It was concluded that X14DesB30 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535, andTA1537) of salmonella typhimurium, and two tryptophan-requiring strains (WP2 pKM101 and WP2 uvrA pKM101) of Escherichia coli, when tested under the onditions employed in this study. The conditions included treatments at concentrations up to 5000 µg/mL, both in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

The present study, NDA report No. T-27, study nr. 940376, is described in a summary study report on Insulin aspart is based on GLP guideline studies prepared by Novo Nordisk. The summarised studies were performed as part of the non-clinical toxicity test regime for authorisation of Insulin aspart as human medicine and the studies are therefore in compliance with the guidelines for authorisation of human medicine.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

other: microtitre fluctuation technique to detect specific gene mutation

Specific details on test material used for the study:

Study performed with the active pharmaceutical ingredient Insulin Aspart

Target gene:

thymidine kinase locus (5-trifluorothymidine restistance) in mouse lymphoma L5178Y cells

Test concentrations with justification for top dose:

Two experiements were performed.

Experiment 1: five doses, seperated by to-fold intervals up to 5000 ug/ml (with and without S-9)

Experiment 2: Five doses rangeing from 1000 to 5000 ug/ml (with and without S-9)

All doses from both experiments were selected to determine viability and 5-trifluorothymidine restistance two days after treatment.

Vehicle / solvent:

Not specified

Details on test system and experimental conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No marked toxicity was observed at any dose of insulin aspart in experiment 1 or 2 in the presence or absence of S-9.
No statistically significant increases in mutant frequency were observed following treatment with insulin aspart at any dose level in the absence or presence of S-9 in either experiment.

Conclusions:

Insulin aspart did not induce a mutagenic reponse in mouse lymphoma cells under the conditions employed in this study.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Due to very close structural similarity between insulin aspart precursor and the read-across source substance Insulin Aspart it is considered justified to perform read-across of mutagenicity data from the source substances to the target substance.
See further read-across justification in document attached in section 13.

Test performed with insulin aspart was negative. Due to the close structural similarity to insulin aspart precursor a lack of mutagenic potential in mammalian cells can be concluded for this substance as well.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Test performed with insulin aspart in mouse lymphoma cells was negative with regard to mutagenic response. Due to the close structural similarity to insulin aspart precursor a lack of mutagenic potential in mammalian cells can be concluded for this substance as well.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: EEC Method B10 specified in the Annex to 92/69/EEC

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory tests (1995)

GLP compliance:

yes

Type of assay:

other: chromosome aberration assay

Specific details on test material used for the study:

MI3, solid, purity of 98.4%

Target gene:

Structural chromosomal aberrations in cultured mammalian cells

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 from rats induces with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1:
14 concentration levels ranging from 118.8 ug/ml and up to 5000 ug/ml

Experiment 2:
12 concentration levels ranging from 118.8 ug/ml and up to 5000 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: Preliminary solubility data indicated that MI3 was soluble in DMSO to a concentration of at least 500 mg/ml.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

Whole blood samples was drawn from the peripheral circulation of 3 non smoking, healthy female volunteers. The volunteers had not knowingly been exposed to high levels of radiation or hazardous chemicals, and have not suffered from viral infections.

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Arochlor 1254 and obtained from Molecular Toxicology Incorporated, USA. The batched of MolTox S-9 were stored frozen in aliquots at -80°C and thawed prior to use. Wach batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens and cytochrome P-450 enzyme activities,

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 48 h
- Exposure duration:
Experiment 1: 20 h
Experiment 2, Pulse treatment: 3 hours, 17 hours recovery

NUMBER OF REPLICATIONS:
4 negative solvent controls (with and without S9)
duplicates of all tests (with and without S9)
2 positive controls (with and without S9)

Evaluation criteria:

100 metaphases from each culture were counted. Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations. Cells with more than 46 chromosomes (polyploid, endoreduplicated and hyperdiploid cells) was recorded separately. Classification of structural aberrations was based on the scheme described by ISCN (12).

A test article is considered as positive if:
1. The proportions of cells with structural aberrations at one or more concentrations exceeds the normal range in both replicates, and
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses

Statistics:

Frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.

Key result

Species / strain:

lymphocytes: Human periferal blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment of cultures with MI3 in the absence and presence of S-9 resulted in frequencies of cells with structural and numerical aberrations which were similar to those in concurrent controls.

Conclusions:

MI3 did not induce chromosome aberrations in cultured peripheral blood lymphocytes when tested up to 5000 µg/mL.

Executive summary:

MI3 was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors. Treatment covering a broad range of doses, separated by narrow intervals, was performed both in the absence and presence of metabolic activation (S-9) at concentrations up to 5000µg/mL. Tests were performed both as continuous exposure for 20 h and with 3 h exposure followed by 17 h recovery exposure. The test article was dissolved in dimethyl sulphoxide.

Treatment of cultures with MI3 in the absence and presence of S-9 resulted in frequencies of cells with aberrations which were similar to this in concurrent negative controls. Number of aberrant cells fell within or close to historical negative control ranges in all treated cultures

It is therefore concluded that MI3 did not induce chromosome aberrations in cultured peripheral blood lymphocytes when tested to 5000µg/mL.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

The present study, NDA report No. T-23, study nr. 940375, is described in a summary study report on Insulin aspart is based on GLP guideline studies prepared by Novo Nordisk. The summarised studies were performed as part of the non-clinical toxicity test regime for authorisation of Insulin aspart as human medicine and the studies are therefore in compliance with the guidelines for authorisation of human medicine.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Studies were conducted with the active pharmaceutical ingredient Insulin Aspart

Target gene:

Not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Histidine requiring

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Histidine requiring

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Histidine requiring

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: Histidine requiring

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: Tryptophan requiring

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Additional strain / cell type characteristics:

other: Tryptophan requiring

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment 1: Five-fold dose interval up to 5000 um/ml
Experiment 2: Two-fold dose interval up to 5000 um/ml

Vehicle / solvent:

No data

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

A treat and plate procedure was used

Rationale for test conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No visible toxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No visible toxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No visible toxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No visible toxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No visible toxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

E. coli, other: WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No visible toxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

N/A

Conclusions:

Under the conditions employed in this study, which included treatments up to 5000 ug/ml both in the absence and in the presense of rat liver metabolic activation system, insulin aspart did not display any mutagenic potential

Executive summary:

Insulin aspart was assayed for mutation in four histidine requiring strains of Salmonella typhimurium and teo tryptophane-requiring strains of Escheria coli. Treatments were conducted with doses up to 5000 ug/ml both in the absence and in the presense of rat liver metabolic activation system. In the present conditions insulin aspart did not display any mutagenic potential

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

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

Species / strain:

S. typhimurium, other: TA 98, 100, 1535 and 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

and WP2 pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Tests performed with insulin aspart; MI3 and X14DesB30 were all negative. Due to the very close structural similarity of these source substances to insulin aspart precursor a lack of mutagenic potential in Salmonella typhimurium and E. coli can be concluded for this substance as well.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The present study, NDA report No. T-24, study nr. 940373, is described in a summary study report on Insulin aspart is based on GLP guideline studies prepared by Novo Nordisk. The summarised studies were performed as part of the non-clinical toxicity test regime for authorisation of Insulin aspart as human medicine and the studies are therefore in compliance with the guidelines for authorisation of human medicine.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: chromosome aberration

Specific details on test material used for the study:

The study was conducted with the active pharmaceutical ingredient Insulin Aspart

Target gene:

not specified

Species / strain / cell type:

lymphocytes: human periferal blood lymphocytes

Details on mammalian cell type (if applicable):

human lymphocyte cell cultures from a male and female donor

Metabolic activation:

with and without

Metabolic activation system:

rat liver post-mitochondrial fraction (S9) from aroclor 1254 induced animals

Test concentrations with justification for top dose:

up to 5000 ug/ml.

Vehicle / solvent:

Not specified

Untreated negative controls:

yes

Remarks:

Histroical negative controls

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Eksperiment 1:
Treatment in the absence of S9 was continuous for 20 hours.

Treatment in the presence of S9 was for 3 hours, followed by 17 hours recovery period prior to harvest.

The test item concentrations for chromosome analysis were selected by evalutating the effect of insulin aspart on mitotic index. Chromosome aberrtions were analysed at three consecutive treatments concentrations up to 5000 ug/ml.


Eksperiment 2:
Treatment in the absence of S9 was continuous for 20 hours.

Treatment in the presence of S9 was for 3 hours, followed by 17 hours recovery period prior to harvest.

A delayed sampling time compared to experiment one of 44 hours and pulse treatment in the absesence of S9 was included in this experiment.

Chromosome aberrations were analysed in celss receiving 20 hour continuous treatments in the absence of S9 and 3 hour pulse treatment in its presence at three consecutive treatment concentrations up to 5000 ug/ml.
The effect of a singe concentration only (without S9) were also investigated at the delayed (44 hour) sampling time after pulse and continuous treatment and following the 3 hour treatment in the absence of S9, at the 20 hour sampling time.

NUMBER OF REPLICATIONS:
2

Rationale for test conditions:

The test item concentrations for chromosome analysis were selected by evalutating the effect of insulin aspart on mitotic index. Chromosome aberrtions were analysed at three consecutive treatments concentrations up to 5000 ug/ml.

Evaluation criteria:

Comparison to historical negative controls and solvent controls.

Statistics:

N/A

Key result

Species / strain:

lymphocytes: Human periferal blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Conclusions:

Insulin aspart did not induce chromosome aberrations in the cultured human periferal blood lymphocytes when tested up to a concentration of 5000 ug/ml in either the absece or presence of S9

Executive summary:

The in vitro clastogenic potential of Insulin aspart was investigated in cultured human peripheral blood lymphocytes. Induction of chromosomal aberrations was investigated in two experiments at concentrations up to 5000 ug/ml with and without metabolic activation by S9. The treated lymphocytes had frequiencies of cells with aberrations which were similar to and not significantly different from those in concurrent solvent controls. Numbers of aberrant cells in all treated cultures fell within historical negative control ranges.

It was therefore concluded that Insulin aspart does not induce chromosome aberrations in cultured human periferal blood lymphocytes when tested up to a concentration of 5000 ug/ml in either the absece or presence of S9.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Key result

Species / strain:

lymphocytes: human lymphocyte cell cultures from a male and female donor

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Tests performed with insulin aspart and MI3 were negative. Due to the very close structural similarity to insulin aspart precursor a lack of chromosome aberration potential in mammalian cells can be concluded for this substance as well.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The conducted in vivo study on insulin aspart does not indicate any genotoxic/clastogenic potential of the substance. Due to very close structural similarity to insulin aspart precursor a lack of genotoxic/ clastogenic potential in vivo can be concluded for this substance as well.

See read-across template and justification attached in section 13

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

unscheduled DNA synthesis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

The present study, NDA report No. T-26, study nr. 940374, is described in a summary study report on Insulin aspart is based on GLP guideline studies prepared by Novo Nordisk. The summarised studies were performed as part of the non-clinical toxicity test regime for authorisation of Insulin aspart as human medicine and the studies are therefore in compliance with the guidelines for authorisation of human medicine.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Study performed using the active pharmaceutical ingredient Insulin aspart as test substance

Species:

rat

Strain:

other: CD rats

Details on species / strain selection:

Not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not specified

Route of administration:

subcutaneous

Vehicle:

Not specified

Details on exposure:

Insulin aspart was administered subcutaneously on two occasions approximately 10 hours apart at a dose volume of 5 ml/kg

Duration of treatment / exposure:

Two injectiions subcutaneusly, approximately 10 hours apart at a dose volume of 5 ml/kg

Frequency of treatment:

Two injections

Post exposure period:

2-4 hours after the second injection (12-14 hours after initial dosing), animals were killed and their liver perfused with collagenase to provide a primary culture of hepatocytes.

Dose / conc.:

2 000 other: U/kg/day

Remarks:

Administered over two doses of 1000 U/kg

Dose / conc.:

800 other: U/kg/day

Remarks:

Administered over two doses

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

yes

Tissues and cell types examined:

hepatocytes from perfused livers

Details of tissue and slide preparation:

Livers were perfused with collagenase to provide primary cultures of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. The net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, for each animal and dose group.

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

N/A

Negative (vehicle) control animals gave a group mean NNG ( net grain count) value of less than zero with only 0.7 -1.7% cells in repair. Group mean NNG values were increased by positive control treatment to at least 7.5%, with more than 50% cells found to be in repair. The vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to a known DNA damaging agent requiring metabolism for its action. The assay was therefore accepted as valid.

Treatment of male and female rats with 800 or 2000 U/kg/day insulin aspart subcutaneously, did not produce a group mean NNG value greater than -1.2, nor were any more than 1% cells found in repair at either dose level.

Conclusions:

Insulin aspart did not to induce unscheduled DNA synsthesis at doses up to 2000 U/kg/day

Executive summary:

Treatment of male and female CD rats with 800 or 2000 U/kg/day insulin aspart subcutaneously, did not produce a group mean NNG value greater than -1.2, nor were any more than 1% cells found in repair at either dose level.

It was therefore concluded that insulin aspart failed to induce unscheduled DNA synsthesis detectable under the experimental conditions employed

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

The present study, NDA report No. T-25, study nr. 940372, is described in a summary study report on Insulin aspart is based on GLP guideline studies prepared by Novo Nordisk. The summarised studies were performed as part of the non-clinical toxicity test regime for authorisation of Insulin aspart as human medicine and the studies are therefore in compliance with the guidelines for authorisation of human medicine.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: micronuclei

Specific details on test material used for the study:

Study performed using the active pharmaceutical ingredient Insulin aspart as test substance

Species:

mouse

Strain:

not specified

Details on species / strain selection:

Not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not specified

Route of administration:

subcutaneous

Vehicle:

Not specified

Details on exposure:

Administration of 200 U/ml on two occasions, 10 hours apart, at a dose volume of 5 ml/kg, allowed a total dose of 2000 U/kg/day to be administered. This corresponds to approximately 2000 times the therapeutic dose, and was selected as the maximum dose administered. In addtion two lower dose levels of 1000 and 500 U/kg/day were administered.

Duration of treatment / exposure:

Two subcutaneous injection, 10 hours apart, over 1 day.

Frequency of treatment:

Ones (two subcutaneous injection, 10 hours apart)

Post exposure period:

Animals were sacrificed 24 or 48 hours after the second treatment.

Dose / conc.:

500 other: U/kg/day

Remarks:

Administered over two injections

Dose / conc.:

1 000 other: U/kg/day

Remarks:

Administered over two injections

Dose / conc.:

2 000 other: U/kg/day

Remarks:

Administered over two injections

No. of animals per sex per dose:

Not specified

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) from bone marrow

Details of tissue and slide preparation:

Not specified

Evaluation criteria:

Micronucleus frequency was estimate din 2000 PCE per animal

Statistics:

Not specified

Key result

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Slides from all dose groups were analysed and micronucleus frequency estimated in 2000 PCE per animal. All positive control animals exhibited increased numbers of micronucleated PCE such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls.
Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE and normal frequencies of micronucleated PCE within historical negative control ranges. Mice treated with insulin aspart at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucelated PCE which were similar to the values for vehicle control groups a both sampling times. There were no instances of statistically significant increases in micornucleus frequency for any of the groups receiving the test article at either sampling time.
The maximum dose caused a marked drop in blood glucose levels in treated mice.

Conclusions:

Insulin aspart did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated with subcutaneous injection using dose levels up to 2000 U/kg/day of inuslin aspart.

Executive summary:

Insulin aspart was teset for its ability to induce micronuclei in the bone marrow of subcutaneously dosed male and female mice. Administration of 2000, 1000 or 500 U/kg/day (divided in two injections) were administered to the mice. Postive and negative (vehicle) control animals were also included. All animals were sacrificed 24 or 48 hours after the second injection.

Slides from all dose groups were analysed and micronucleus frequency estimated in 2000 PCE per animal. All positive control animals exhibited increased numbers of micronucleated PCE such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls.

Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE and normal frequencies of micronucleated PCE within historical negative control ranges. Mice treated with insulin aspart at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucelated PCE which were similar to the values for vehicle control groups a both sampling times. There were no instances of statistically significant increases in micornucleus frequency for any of the groups receiving the test article at either sampling time.

It was concluded that insulin aspart did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 U/kg/day.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Due to very close structural similarity between insulin aspart precursor and the read-across source substance, insulin aspart, and due to similar physico-chemical properties it is considered justified to perform read-across of in vivo mutagenicity data from the source substance to the target substance.
see further read-across justification in document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Conclusions:

Test performed with insulin aspart was negative with respect to micronuclei formation in erythrocytes from mice exposured subcutaneously. Due to very close structural similarity to insulin aspart precursor a lack of clastogenic potential in mammalian cells can be concluded for this substance as well.

Reference 4

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Due to very close structural similarity between insulin aspart precursor and the read-across source substance, insulin aspart, and due to similar physico-chemical properties it is considered justified to perform read-across of in vivo mutagenicity data from the source substance to the target substance.
See further read-across justification in document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Conclusions:

Test performed with insulin aspart using subcutaneous administrat in rats was negative with regard to induction of unscheduled DNA synthesis. Due to the close structural similarity to insulin aspart precursor a lack of genotoxic potential in mammalian cells in vivo can be concluded for this substance as well.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on a consistent pattern of negative test results in in vitro mutagenicity testing of the three read-across substances inuslin aspart, MI3 and X14DeB30 and negative results in in vivo testing of insulin aspart a lack of genotoxic potential can be concluded for insulin aspart precurtsor as well. Thus, no classification for mutagenicity according to the CLP-criteria applies.