Insulin Aspart Precursor

Endpoint summary

Description of key information

The oral administration of MI3 at dose levels of 0, 100, 300, 1000 mg/kg/d to Wistar rats produced no evidence of toxicity and no sign of effects could be attributed to the treatment. I.e. due to structural similarity to insulin aspart precusor the oral NOAEL of 1000 mg/kg/d is considered relevant for this substance as well.

In a 26 week inhalation study female and male rats were subjected to 0.5 h of daily inhalation exposure to insulin aspart aerosols in the concentration range of 344 – 2074 mg/m³for 26 weeks. Increased female mortality was observed within the first 4 weeks at a concentration level of 1796 mg/m³. The lethal outcome was found to be due to severe hypoglycaemia. Such effects were not found at an exposure level of 805 mg/m3 that can be considered as a NOAEC. This NOAEC that is in connection with the substance insulin aspart (an active pharmaceutical ingredient) that bind to the inuslin receptor may be used as a very conservative NOAEC for inuslin aspart precusor also, although a considerable higher NOAEC value would be expected for a structural similar substance not leading to a hypoglycaemic response.

See read-across template and justification attached in section 13

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

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

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

Based on read across to MI3 (S2), the NOEL for repeated dose toxicity after oral exposure is set to 1000 mg/kg/d

Executive summary:

MI3 was adinistration to Han Wistar rats at doses of 100, 300 or 1000 mg/kg/day for 28 days. No evidence of toxicity and no effects which could be confidently attributed to treatment was observed. Due to the very close structural similarity to of MI3 to insulin aspart precursor these results can be used for read acoss purposes and the NOEL for repeated dose toxicity after oral exposure is set to 1000 mg/kg/d.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

march to december 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C202483
- Expiration date of the lot/batch: 31.05.2001
- Purity: 98.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -10 degrees C

- Solubility and stability of the test substance in the solvent/vehicle:
MI3 was homogeneous and stable in purified water for up to 6 hours after dose preparation.

Species:

rat

Strain:

other: Han Wistar rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 193,8-228,9 g; females: 122,5-175 g
- Fasting period before study: not specified
- Housing: The animals were housed in a single, exclusive room, airconditioned (15 air changes/hour)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water

Details on oral exposure:

Oral exposure by gavage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulations for achieved concentration were performed in week 1. The analysis was performed by Covance and demonstrated that the formulations were accurately performed.

Duration of treatment / exposure:

29 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Controls

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

intermediate dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

N/A

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily observation for signs of ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

Post dosing observations were performed daily during the first week of treatment (excluding Day 2, as functional obervational battery was being performed) at the following time intervals: immediately after dosing, 30 minutes, 1, 2 and 4 hours after dosing. For the remainder of the study, post dosing observations were recorded twice weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Before treatment in the first day of dosing, at weekly intervals and before necropsy

FOOD CONSUMPTION:
- Food consumption was determined weekly and calculated as g food/animal/week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of week 4
- Anaesthetic used for blood collection: Yes (fluothane)
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes overnight
- How many animals: all animals
- Parameters examined:
haemoglobin concentration
packed cell volume
mean cell haemoglobin
reticulocytes
platelet couunt
red blood cell count
mean cell volume
mean cell haemoglobin concentration
total and differential white cell count
and prothromnin time (in trisodium citrate anticoagulant)

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes: FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
- Time schedule for examinations: before initiation of treatment and once weekly thereafter.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity (week 4)

IMMUNOLOGY:No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes on control and high dose animals


HISTOPATHOLOGY: Yes on control and high dose animals

Statistics:

Statistical analysis were perforned where appropriate.
Body weight gains, necropsy body weight, haematology, clinical chemistry, locomotor and FOB data were analysed using two-way analysis of variance (ANOVA). Pairwise comparison with control for each sex separately, were made using Dunnett’s test. For each sex separately, a regression test was performed to determine whether there was a dose response relationship. Non-parametric methods were used for clinical parameters with values above or below limit of detection. Levene’s test for equality of variance across groups, sexes and any interactions was also performed.
Organ weights were analysed using analysis of covariance (ANCOVA) and Dunnett’s test.
Significance level of p>0.05 was used. (p<0.01 for Levene's test)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Total group mean motor activity for females given 1000 mg/kg/day was slightly lower than controls. Landing foot display for males given 300 or 1000 mg/kg/day were slightly higher than controls.
Individual results for both landing footsplay and motor activity for the control and the treated groups were generally variable. This slight changes in footsplay and motor activity are therefore not considered to be related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Liver weight for males given 1000 mg/kg/day and brain weight for females given 1000 mg/kg/day were slightly lower than controls, however, there were no clear sex or dosage-relationships. These findings are therefore considered to be fortuitous.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The administration of MI3 at doses of 100, 300 or 1000 mg/kg/day to Han Wistar rats for 28 days produced no evidence of toxicity and no effects which could be confidently attributed to treatment.

Executive summary:

MI3 was administered to Han Wistar rats at doses of 100, 300 or 1000 mg/kg/day for 28 days. No evidence of toxicity and no effects which could be confidently attributed to treatment was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2007 - April 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: European Parliament and Council Directive 2001/83/EC of 6 November 2001 of the Community Code Relating to Medicinal Products for Human Use, OJ L311/67-128, 28 November 2001 as amended Commission Directive 2003/63/EC, OJ L159, 27 June 2003.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The active phamacautical ingredient Insulin aspart. Micronised powder. Purity > 98.6%

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of toxicological
changes in man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD)
strain was used because of the historical control data available in this laboratory and because
previous studies with the test articles have been performed in this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Male and female Crl:CD(SD) rats were received from Charles River (UK) Ltd.
The rats were ordered to be 42 to 49 days of age for males and 49 to 56 days of age for females on
arrival and within at a minimum weight of 225 g for males and 175 g for females.

On arrival, the animals were removed from the transit boxes and allocated to study cages. Using
the sequence of cages in the battery, one animal at a time was placed in each cage with the
procedure being repeated until each cage held the appropriate number of animals. Each sex was
allocated separately.

Each animal was assigned a number and identified uniquely within the study by a tail tattoo. Each
cage label was colour-coded according to group and was numbered uniquely with cage and study
number, as well as the identity of the occupants.

The animals were allowed to acclimatise to the conditions described below for 10 days before
treatment commenced. For those animals selected for this study, their age at the start of treatment
was 52 to 59 days for males and 59 to 66 days for females and their bodyweights were in the range
of 286 to 416 g for males and 193 to 265 g for females.
The spare animals were sacrificed after treatment commenced.

The animals were housed five of one sex per cage for the Main Study and three of one sex per cage
for the Recovery and Satellite Study, unless this number was reduced by mortality or isolation. The
cages had solid polypropylene bottoms and stainless steel mesh lids. Cages, food hoppers and
water bottles were changed at appropriate intervals.

DIET AND WATER:
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance
Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with
sipper tubes.

Each animal was provided with an Aspen chew block for environmental enrichment. Chew blocks
were provided throughout the study and were replaced when necessary, but at a minimum
frequency of every two weeks.

Each batch of diet was analysed routinely by the supplier for various nutritional components and
chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and
approved before any batch of diet was released for use. The quality of the water supply is governed
by regulations published by the Department for Environment, Food and Rural Affairs. Certificates
of analysis were received routinely from the water supplier. Certificates of analysis were received
routinely from the supplier of the aspen chew blocks. Since the results of these various analyses did
not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were
analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

ENVIRONMENTAL CONDITIONS:
Animals were housed inside a restricted entry rodent facility (Building U20, Room 003). The
facility was designed and operated to minimise the entry of external biological and chemical agents
and to minimise the transfer of such agents between rooms. Before the study the room was cleaned
and disinfected with a bactericide.

The temperature and relative humidity controls were maintained within the range of 19 to 23°C and
40 to 70% respectively. Artificial lighting was controlled to give a cycle of 12 hours continuous
light and 12 hours continuous dark per 24 hours, with simulated periods of dawn and dusk.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and
humidity were monitored continuously. Since these data show that there were no significant
deviations from target values, they are not presented. Alarms were activated if there was any failure
of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available
to be automatically brought into operation should the public supply fail.

The animals were housed five of one sex per cage for the Main Study and three of one sex per cage
for the Recovery and Satellite Study, unless this number was reduced by mortality or isolation. The
cages had solid polypropylene bottoms and stainless steel mesh lids. Cages, food hoppers and
water bottles were changed at appropriate intervals.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 0.26 - < 3.42 µm

Geometric standard deviation (GSD):

1.6

Remarks on MMAD:

The mean respirable fraction (% < 7 μm aerodynamic diameter) was greater than or equal to 95% for all groups.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Overall the mean achieved aerosol concentrations for Insulin Aspart were 95, 89 and 91% of
target for Groups 2, 3 and 4 male, respectively. The achieved concentration of Insulin Aspart for
Group 4 females was 79% of a target concentration of 59.7 U/L (Weeks 1-6 of treatment), and
99% of a target concentration of 31.8 U/L (Weeks 6-26 of treatment), resulting in an overall
concentration of 35.1 U/L, calculated as a time weighted average.

Duration of treatment / exposure:

30 min/day for 26 weeks

Frequency of treatment:

Daily

Dose / conc.:

344 mg/m³ air (analytical)

Remarks:

corresponding to 9.07 U/L (males and females)

Dose / conc.:

805 mg/m³ air (analytical)

Remarks:

corresponding to 21.2 U/L (males and females)

Dose / conc.:

2 074 mg/m³ air (analytical)

Remarks:

corresponding to 54.6 U/L (males)

Dose / conc.:

1 796 mg/m³ air (analytical)

Remarks:

corresponding to 47.3 U/L (week 1-6, females)

Dose / conc.:

1 200 mg/m³ air (analytical)

Remarks:

corresponding to 31.6 U/L (week 7-26, females)

No. of animals per sex per dose:

20 animals per sex per dose group in the core study.
Additionally 6 male and 6 female recovery animals were assigned to the highest dose Insulin Aspart
to assess recovery from any treatment related effect.
Further 12 males and/or 12 females were assigned to each group and used for antibody
analysis, blood glucose measurements and toxicokinetic evaluation.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed clinical observations were recorded as “dosing signs” at the following times in relation to dose administration:
Immediately before dosing
During and immediately after dosing
Between half an hour and two hours after completion of dosing
As late as possible in the working day.

In addition, a more detailed weekly physical examination labelled as “clinical signs” was performed on each animal to monitor general health as each animal was taken out of its cage specifically. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.On days on which blood glucose measurements were not scheduled the animals were also observed without removal from the cage, half-hourly for 3 hours following dosing for signs of hypoglycaemia.

BODY WEIGHT: Yes
- Time schedule for examinations:
The weight of each rat was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), at weekly intervals throughout the treatment and recovery periods, and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and at weekly intervals throughout the treatment and recovery periods. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, the eyes of all main and recovery animals allocated to the study (including spare animals) were examined by means of a binocular indirect ophthalmoscope. During Week 26 of treatment the eyes of all main and recovery animals. The examination was extended to the Week 4 of the recovery phase for recovery animals of Groups 4M, 4F, 6 and 7.

- Dose groups that were examined: All main and recovery animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 13 and 26 of treatment (before dosing on each occasion) and Week 4 of recovery
- Anaesthetic used for blood collection: Yes Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the retro-orbital sinus.
- Animals fasted: No
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals: all main and recovery animals
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 13/14 (males and females respectively) and Week 26 of treatment and at Week 4 of the recovery phase
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: MORTALITY:
Debilitated animals were observed carefully and, where necessary, isolated to prevent cannibalism. Animals judged in extremis were killed to prevent unnecessary or prolonged suffering. Where possible, blood samples were taken ante mortem and analysed for the parameters specified in the haematology and blood chemistry sections below. A complete necropsy was performed in all cases

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

Summary statistics (e.g. means and standard deviations) were calculated from computer-stored individual raw data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 117, male animal 48 at 805 mg/m3 suffered a convulsion coupled with unresponsive behaviour.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 15 exposed animals died, prior to scheduled termination.
There were 12 deaths in the main study and recovery animals at which 9 occurred at the highest dose level. 6 females died at the highest dose within the first 6 weeks and 3 wihtin the first 4 weeks.
level 6 died. The incidence of deaths at the two lower exposure levels (n=2+1) of Insulin Aspart was not clearly greater than that in controls (n=1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight reduction in mean bodyweight gain was observed following 26 weeks of treatment in all
treated groups compared with concurrent controls (up to 0.86 x controls).

Between Weeks 1 and 4 of recovery, all groups showed an increased mean bodyweight gain at a
rate higher than during the treatment phase.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The variability observed for several haematological parameters, showed no consistency between the
sexes or the occasions and were often due to a greater individual variability in the treated or control
groups.
Following 4 weeks of recovery, there were no observed differences in the haematological
parameters compared with during treatment

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Tmax of insulin aspart was observed between 0.25 and 1 hour post exposure.
There was a general treatment related decrease in blood glucose levels in both male and female
animals exposed to Insulin Aspart on Day 1 and at Week 26.

Mean bilirubin levels were statistically significantly increased in females at the two highest dose levels
during Week 26 of treatment (1.6X and 1.5X control respectively).

There were no other findings considered to be attributable to treatment. Significant differences were apparent in other parameters, but due to the lack of consistency between the sexes or the occasions and individual variability in the treated or control groups, they were considered to be unrelated with either treatment.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher urine volume was apparent in Weeks 13 and 26 for males at 2074 mg/m3 and in Week 26 for males at 805 mg/m3.
There were no other changes considered to be treatment related effects for animals during exposure to Insulin Aspart or Human Insulin or at the end of the recovery period.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after 26 weeks of treatment revealed the following
changes in the lungs.

A slightly increased incidence of dark foci was observed in male rats compared with controls. Due to the poor dose relationship and absence of significant histological changes it is not considered treatment related.

The nature and incidence of all other findings were consistent with the commonly seen background
of macroscopic changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Variable numbers of animals in all groups showed epithelial hyperplasia of the arytenoids in the
larynx, this was also seen in control animals. Due to lack of a dose relationship and this finding
being a variable feature also in controls, this is considered to be background.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

805 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other:

Remarks:

Based on hypoglycaemia associated deaths

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

156 mg/m³ air (analytical)

System:

other: Blood sugar regulation

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on malfunction leading to death due to hypoglycaemia, the No Observed Adverse Effect Concentration evel (NOAEC) for Insulin Aspart was 805 mg/m3.

Executive summary:

Insulin Aspart was administered to CD rats by snout-only inhalation exposure for 30 minutes per day for 26 weeks at achieved concentration levels of:

Females:   0; 344; 805; 1796 mg/m³(week 1-6) + 1200 mg/m³(week 7-26)

 Males: 0; 344; 805; 2074 mg/m³

There were no treatment-related effects on clinical signs, food consumption, ophthalmoscopy, haematology (peripheral and bone marrow), organ weights or microscopic pathology findings.

All observed treatment-related effects were considered to be attributable to the pharmacological effect of insulin on plasma glucose concentrations. The initial dose level of 1796 mg/m³Insulin Aspart was not tolerated by female rats as plasma glucose concentrations decreased following dosing and deaths occurred due to hypoglycaemia. The reduced dose of 1200 mg/m³for continuation of the stady was well tolerated for 19½ weeks. In males exposure to 1796 mg/m³resulted in a small number of potentially hypoglycaemia-related deaths.

Based on the hypoglycaemia associated deaths, the No Observed Adverse Effect Concentration (NOAEC) for Insulin Aspart was 805 mg/m³.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

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 substances: insulin aspart and due to similar physico-chemical properties and similar potential for absorption and systemic uptake from oral, dermal and inhalational exposure it is considered justified to make use of read-across of toxicological data from the source substances to the target substance.

The primary function of human insulin (and insulin aspart) is regulation of glucose metabolism, as insulins bind to the insulin receptors on muscle and fat cells and lower blood glucose by facilitating the cellular uptake of glucose and simultaneously inhibiting the output of glucose from the liver. Thus, exposure to substances acting as insulin induce a hypoglycaemic response.
However, in the target substance the two polypeptide subunits in the insulin-like structure are linked together resulting in a change in the steric configuration of the molecule and thus, the substance cannot bind to the insulin receptor and therefore cannot induce an insulin-like hypoglycaemic response. Thus, it would only be appropirate to make read-across on effects not linked to an insulin-like response.

In the S1-study (insulin aspart), female and male rats were subjected to 0.5 h of daily inhalation exposure to insulin aspart aerosols in the concentration range of 344 – 2074 mg/m3 for 26 weeks. Increased female mortality was observed within the first 4 weeks at a concentration level of 1796 mg/m3. The lethal outcome were found to be due to severe hypoglycaemia. In this study a NOAEL of 805 mg/m3 was established based on hypoglycemic induced deaths.
As no effects not linked to a hypoglycaemic response was noted a cautious approach for read-across would be to consider this NOAEC as a NOAEC for insulin aspart precusor as well even though insulin aspart precusor may be considered as less toxic as no pharmacological insulin like response is expected from this substance.

Thus, a NOAEC of 805 mg/m3 for daily ½ hour inhalation exposure can be established for inuslin aspart precusor in relation to a 26 week exposure period in rats.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEC

Effect level:

805 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other:

Remarks:

Based on hypoglycaemia associated deaths

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 796 mg/m³ air (analytical)

System:

other: blood sugar regulation

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on read across to Insulin aspart (S1), the NOAEC of 805 mg/m3 for daily ½ hour inhalation exposure can be established for in relation to a 26 week exposure period in rats.

Executive summary:

Due to very close structural similarity between insulin aspart precursor and the read-across source substances: insulin aspart and due to similar physico-chemical properties and similar potential for absorption and systemic uptake from oral, dermal and inhalational exposure it is considered justified to make use of read-across of toxicological data from the source substances to the target substance.  

The primary function of human insulin (and insulin aspart) is regulation of glucose metabolism, as insulins bind to the insulin receptors on muscle and fat cells and lower blood glucose by facilitating the cellular uptake of glucose and simultaneously inhibiting the output of glucose from the liver. Thus, exposure to substances acting as insulin induce a hypoglycaemic response. However, in the target substance the two polypeptide subunits in the insulin-like structure are linked together resulting in a change in the steric configuration of the molecule and thus, the substance cannot bind to the insulin receptor and therefore cannot induce an insulin-like hypoglycaemic response. Thus, it would only be appropirate to make read-across on effects not mediated via an insulin-like response.

In the S1-study (insulin aspart), female and male rats were subjected to 0.5 h of daily inhalation exposure to insulin aspart aerosols in the concentration range of 344 – 2074 mg/m3 for 26 weeks. Increased female mortality was observed within the first 4 weeks at a concentration level of 1796 mg/m3. The lethal outcome were found to be due to severe hypoglycaemia. In this study a NOAEC of 805 mg/m3 was established based on hypoglycemic induced deaths.

A cautious approach for read-across would be to consider this NOAEC as a NOAEC for insulin aspart precusor as well even though insulin aspart precusor may be considered as less toxic as no pharmacological response is expected from this substance.

Thus, a NOAEC of 805 mg/m3 for daily ½ hour inhalation exposure can be established for inuslin aspart precusor in relation to a 26 week exposure period in rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

805 mg/m³

Study duration:

subchronic

Species:

rat

System:

other: Mortality

Organ:

not specified

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2007 - April 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: European Parliament and Council Directive 2001/83/EC of 6 November 2001 of the Community Code Relating to Medicinal Products for Human Use, OJ L311/67-128, 28 November 2001 as amended Commission Directive 2003/63/EC, OJ L159, 27 June 2003.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The active phamacautical ingredient Insulin aspart. Micronised powder. Purity > 98.6%

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of toxicological
changes in man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD)
strain was used because of the historical control data available in this laboratory and because
previous studies with the test articles have been performed in this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Male and female Crl:CD(SD) rats were received from Charles River (UK) Ltd.
The rats were ordered to be 42 to 49 days of age for males and 49 to 56 days of age for females on
arrival and within at a minimum weight of 225 g for males and 175 g for females.

On arrival, the animals were removed from the transit boxes and allocated to study cages. Using
the sequence of cages in the battery, one animal at a time was placed in each cage with the
procedure being repeated until each cage held the appropriate number of animals. Each sex was
allocated separately.

Each animal was assigned a number and identified uniquely within the study by a tail tattoo. Each
cage label was colour-coded according to group and was numbered uniquely with cage and study
number, as well as the identity of the occupants.

The animals were allowed to acclimatise to the conditions described below for 10 days before
treatment commenced. For those animals selected for this study, their age at the start of treatment
was 52 to 59 days for males and 59 to 66 days for females and their bodyweights were in the range
of 286 to 416 g for males and 193 to 265 g for females.
The spare animals were sacrificed after treatment commenced.

The animals were housed five of one sex per cage for the Main Study and three of one sex per cage
for the Recovery and Satellite Study, unless this number was reduced by mortality or isolation. The
cages had solid polypropylene bottoms and stainless steel mesh lids. Cages, food hoppers and
water bottles were changed at appropriate intervals.

DIET AND WATER:
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance
Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with
sipper tubes.

Each animal was provided with an Aspen chew block for environmental enrichment. Chew blocks
were provided throughout the study and were replaced when necessary, but at a minimum
frequency of every two weeks.

Each batch of diet was analysed routinely by the supplier for various nutritional components and
chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and
approved before any batch of diet was released for use. The quality of the water supply is governed
by regulations published by the Department for Environment, Food and Rural Affairs. Certificates
of analysis were received routinely from the water supplier. Certificates of analysis were received
routinely from the supplier of the aspen chew blocks. Since the results of these various analyses did
not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were
analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

ENVIRONMENTAL CONDITIONS:
Animals were housed inside a restricted entry rodent facility (Building U20, Room 003). The
facility was designed and operated to minimise the entry of external biological and chemical agents
and to minimise the transfer of such agents between rooms. Before the study the room was cleaned
and disinfected with a bactericide.

The temperature and relative humidity controls were maintained within the range of 19 to 23°C and
40 to 70% respectively. Artificial lighting was controlled to give a cycle of 12 hours continuous
light and 12 hours continuous dark per 24 hours, with simulated periods of dawn and dusk.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and
humidity were monitored continuously. Since these data show that there were no significant
deviations from target values, they are not presented. Alarms were activated if there was any failure
of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available
to be automatically brought into operation should the public supply fail.

The animals were housed five of one sex per cage for the Main Study and three of one sex per cage
for the Recovery and Satellite Study, unless this number was reduced by mortality or isolation. The
cages had solid polypropylene bottoms and stainless steel mesh lids. Cages, food hoppers and
water bottles were changed at appropriate intervals.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 0.26 - < 3.42 µm

Geometric standard deviation (GSD):

1.6

Remarks on MMAD:

The mean respirable fraction (% < 7 μm aerodynamic diameter) was greater than or equal to 95% for all groups.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Overall the mean achieved aerosol concentrations for Insulin Aspart were 95, 89 and 91% of
target for Groups 2, 3 and 4 male, respectively. The achieved concentration of Insulin Aspart for
Group 4 females was 79% of a target concentration of 59.7 U/L (Weeks 1-6 of treatment), and
99% of a target concentration of 31.8 U/L (Weeks 6-26 of treatment), resulting in an overall
concentration of 35.1 U/L, calculated as a time weighted average.

Duration of treatment / exposure:

30 min/day for 26 weeks

Frequency of treatment:

Daily

Dose / conc.:

344 mg/m³ air (analytical)

Remarks:

corresponding to 9.07 U/L (males and females)

Dose / conc.:

805 mg/m³ air (analytical)

Remarks:

corresponding to 21.2 U/L (males and females)

Dose / conc.:

2 074 mg/m³ air (analytical)

Remarks:

corresponding to 54.6 U/L (males)

Dose / conc.:

1 796 mg/m³ air (analytical)

Remarks:

corresponding to 47.3 U/L (week 1-6, females)

Dose / conc.:

1 200 mg/m³ air (analytical)

Remarks:

corresponding to 31.6 U/L (week 7-26, females)

No. of animals per sex per dose:

20 animals per sex per dose group in the core study.
Additionally 6 male and 6 female recovery animals were assigned to the highest dose Insulin Aspart
to assess recovery from any treatment related effect.
Further 12 males and/or 12 females were assigned to each group and used for antibody
analysis, blood glucose measurements and toxicokinetic evaluation.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed clinical observations were recorded as “dosing signs” at the following times in relation to dose administration:
Immediately before dosing
During and immediately after dosing
Between half an hour and two hours after completion of dosing
As late as possible in the working day.

In addition, a more detailed weekly physical examination labelled as “clinical signs” was performed on each animal to monitor general health as each animal was taken out of its cage specifically. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.On days on which blood glucose measurements were not scheduled the animals were also observed without removal from the cage, half-hourly for 3 hours following dosing for signs of hypoglycaemia.

BODY WEIGHT: Yes
- Time schedule for examinations:
The weight of each rat was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), at weekly intervals throughout the treatment and recovery periods, and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and at weekly intervals throughout the treatment and recovery periods. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, the eyes of all main and recovery animals allocated to the study (including spare animals) were examined by means of a binocular indirect ophthalmoscope. During Week 26 of treatment the eyes of all main and recovery animals. The examination was extended to the Week 4 of the recovery phase for recovery animals of Groups 4M, 4F, 6 and 7.

- Dose groups that were examined: All main and recovery animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 13 and 26 of treatment (before dosing on each occasion) and Week 4 of recovery
- Anaesthetic used for blood collection: Yes Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the retro-orbital sinus.
- Animals fasted: No
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals: all main and recovery animals
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 13/14 (males and females respectively) and Week 26 of treatment and at Week 4 of the recovery phase
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: MORTALITY:
Debilitated animals were observed carefully and, where necessary, isolated to prevent cannibalism. Animals judged in extremis were killed to prevent unnecessary or prolonged suffering. Where possible, blood samples were taken ante mortem and analysed for the parameters specified in the haematology and blood chemistry sections below. A complete necropsy was performed in all cases

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

Summary statistics (e.g. means and standard deviations) were calculated from computer-stored individual raw data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 117, male animal 48 at 805 mg/m3 suffered a convulsion coupled with unresponsive behaviour.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 15 exposed animals died, prior to scheduled termination.
There were 12 deaths in the main study and recovery animals at which 9 occurred at the highest dose level. 6 females died at the highest dose within the first 6 weeks and 3 wihtin the first 4 weeks.
level 6 died. The incidence of deaths at the two lower exposure levels (n=2+1) of Insulin Aspart was not clearly greater than that in controls (n=1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight reduction in mean bodyweight gain was observed following 26 weeks of treatment in all
treated groups compared with concurrent controls (up to 0.86 x controls).

Between Weeks 1 and 4 of recovery, all groups showed an increased mean bodyweight gain at a
rate higher than during the treatment phase.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The variability observed for several haematological parameters, showed no consistency between the
sexes or the occasions and were often due to a greater individual variability in the treated or control
groups.
Following 4 weeks of recovery, there were no observed differences in the haematological
parameters compared with during treatment

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Tmax of insulin aspart was observed between 0.25 and 1 hour post exposure.
There was a general treatment related decrease in blood glucose levels in both male and female
animals exposed to Insulin Aspart on Day 1 and at Week 26.

Mean bilirubin levels were statistically significantly increased in females at the two highest dose levels
during Week 26 of treatment (1.6X and 1.5X control respectively).

There were no other findings considered to be attributable to treatment. Significant differences were apparent in other parameters, but due to the lack of consistency between the sexes or the occasions and individual variability in the treated or control groups, they were considered to be unrelated with either treatment.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher urine volume was apparent in Weeks 13 and 26 for males at 2074 mg/m3 and in Week 26 for males at 805 mg/m3.
There were no other changes considered to be treatment related effects for animals during exposure to Insulin Aspart or Human Insulin or at the end of the recovery period.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after 26 weeks of treatment revealed the following
changes in the lungs.

A slightly increased incidence of dark foci was observed in male rats compared with controls. Due to the poor dose relationship and absence of significant histological changes it is not considered treatment related.

The nature and incidence of all other findings were consistent with the commonly seen background
of macroscopic changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Variable numbers of animals in all groups showed epithelial hyperplasia of the arytenoids in the
larynx, this was also seen in control animals. Due to lack of a dose relationship and this finding
being a variable feature also in controls, this is considered to be background.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

805 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other:

Remarks:

Based on hypoglycaemia associated deaths

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

156 mg/m³ air (analytical)

System:

other: Blood sugar regulation

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on malfunction leading to death due to hypoglycaemia, the No Observed Adverse Effect Concentration evel (NOAEC) for Insulin Aspart was 805 mg/m3.

Executive summary:

Insulin Aspart was administered to CD rats by snout-only inhalation exposure for 30 minutes per day for 26 weeks at achieved concentration levels of:

Females:   0; 344; 805; 1796 mg/m³(week 1-6) + 1200 mg/m³(week 7-26)

 Males: 0; 344; 805; 2074 mg/m³

There were no treatment-related effects on clinical signs, food consumption, ophthalmoscopy, haematology (peripheral and bone marrow), organ weights or microscopic pathology findings.

All observed treatment-related effects were considered to be attributable to the pharmacological effect of insulin on plasma glucose concentrations. The initial dose level of 1796 mg/m³Insulin Aspart was not tolerated by female rats as plasma glucose concentrations decreased following dosing and deaths occurred due to hypoglycaemia. The reduced dose of 1200 mg/m³for continuation of the stady was well tolerated for 19½ weeks. In males exposure to 1796 mg/m³resulted in a small number of potentially hypoglycaemia-related deaths.

Based on the hypoglycaemia associated deaths, the No Observed Adverse Effect Concentration (NOAEC) for Insulin Aspart was 805 mg/m³.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

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 substances: insulin aspart and due to similar physico-chemical properties and similar potential for absorption and systemic uptake from oral, dermal and inhalational exposure it is considered justified to make use of read-across of toxicological data from the source substances to the target substance.

The primary function of human insulin (and insulin aspart) is regulation of glucose metabolism, as insulins bind to the insulin receptors on muscle and fat cells and lower blood glucose by facilitating the cellular uptake of glucose and simultaneously inhibiting the output of glucose from the liver. Thus, exposure to substances acting as insulin induce a hypoglycaemic response.
However, in the target substance the two polypeptide subunits in the insulin-like structure are linked together resulting in a change in the steric configuration of the molecule and thus, the substance cannot bind to the insulin receptor and therefore cannot induce an insulin-like hypoglycaemic response. Thus, it would only be appropirate to make read-across on effects not linked to an insulin-like response.

In the S1-study (insulin aspart), female and male rats were subjected to 0.5 h of daily inhalation exposure to insulin aspart aerosols in the concentration range of 344 – 2074 mg/m3 for 26 weeks. Increased female mortality was observed within the first 4 weeks at a concentration level of 1796 mg/m3. The lethal outcome were found to be due to severe hypoglycaemia. In this study a NOAEL of 805 mg/m3 was established based on hypoglycemic induced deaths.
As no effects not linked to a hypoglycaemic response was noted a cautious approach for read-across would be to consider this NOAEC as a NOAEC for insulin aspart precusor as well even though insulin aspart precusor may be considered as less toxic as no pharmacological insulin like response is expected from this substance.

Thus, a NOAEC of 805 mg/m3 for daily ½ hour inhalation exposure can be established for inuslin aspart precusor in relation to a 26 week exposure period in rats.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEC

Effect level:

805 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other:

Remarks:

Based on hypoglycaemia associated deaths

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 796 mg/m³ air (analytical)

System:

other: blood sugar regulation

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on read across to Insulin aspart (S1), the NOAEC of 805 mg/m3 for daily ½ hour inhalation exposure can be established for in relation to a 26 week exposure period in rats.

Executive summary:

Due to very close structural similarity between insulin aspart precursor and the read-across source substances: insulin aspart and due to similar physico-chemical properties and similar potential for absorption and systemic uptake from oral, dermal and inhalational exposure it is considered justified to make use of read-across of toxicological data from the source substances to the target substance.  

The primary function of human insulin (and insulin aspart) is regulation of glucose metabolism, as insulins bind to the insulin receptors on muscle and fat cells and lower blood glucose by facilitating the cellular uptake of glucose and simultaneously inhibiting the output of glucose from the liver. Thus, exposure to substances acting as insulin induce a hypoglycaemic response. However, in the target substance the two polypeptide subunits in the insulin-like structure are linked together resulting in a change in the steric configuration of the molecule and thus, the substance cannot bind to the insulin receptor and therefore cannot induce an insulin-like hypoglycaemic response. Thus, it would only be appropirate to make read-across on effects not mediated via an insulin-like response.

In the S1-study (insulin aspart), female and male rats were subjected to 0.5 h of daily inhalation exposure to insulin aspart aerosols in the concentration range of 344 – 2074 mg/m3 for 26 weeks. Increased female mortality was observed within the first 4 weeks at a concentration level of 1796 mg/m3. The lethal outcome were found to be due to severe hypoglycaemia. In this study a NOAEC of 805 mg/m3 was established based on hypoglycemic induced deaths.

A cautious approach for read-across would be to consider this NOAEC as a NOAEC for insulin aspart precusor as well even though insulin aspart precusor may be considered as less toxic as no pharmacological response is expected from this substance.

Thus, a NOAEC of 805 mg/m3 for daily ½ hour inhalation exposure can be established for inuslin aspart precusor in relation to a 26 week exposure period in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

Based on read-across and evidence that indicte that insulin aspart precusor does not induce hypoglycaemia, insulin aspart precusor should not be classified with STOT RE according to the CLP-criteria.