Insulin Aspart Precursor

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline (1997)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: Cream powder (84% w/w)

Specific details on test material used for the study:

MI3, solid, purity of 98.4%

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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)