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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2007 - April 208
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Human Insulin
Molecular formula:
C257H383N65O77S6
IUPAC Name:
Human Insulin
Test material form:
solid: particulate/powder
Remarks:
White powder
Details on test material:
Molecular formula: C257H383N65O77S6
Molecular weight: 5807.66 g/mol
Specific details on test material used for the study:
The study was also performed with Insulin Aspart

Test animals

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.

Administration / exposure

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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Overall the mean achieved aerosol concentrations for Human Insulin were 93, 96 and 109% of the target concentrations for the three groups, respectively.

For Insulin Aspart 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 (Weeks 1-6 of treatment), and 99% of a target concentration (Weeks 6-26 of treatment)
Duration of treatment / exposure:
30 min/day for 26 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/m³ air (analytical)
Remarks:
Controls (males and females)
Dose / conc.:
847 mg/m³ air (analytical)
Remarks:
Human insulin, corresponding to 22.3 U/L (males and females)
Dose / conc.:
1 162 mg/m³ air (analytical)
Remarks:
Human insulin, corresponding to 0.6 U/L (females)
Dose / conc.:
1 979 mg/m³ air (analytical)
Remarks:
Human insulin, corresponding to 37 U/L (males)
Dose / conc.:
344 mg/m³ air (analytical)
Remarks:
Insulin aspart, corresponding to 9.07 U/L (males and females)
Dose / conc.:
805 mg/m³ air (analytical)
Remarks:
Insulin aspart, corresponding to 21.2 U/L (males and females)
Dose / conc.:
2 074 mg/m³ air (analytical)
Remarks:
Insulin aspart, corresponding to 54.6 U/L (males)
Dose / conc.:
1 796 mg/m³ air (analytical)
Remarks:
Insulin aspart, corresponding to 47.3 U/L (females)
Dose / conc.:
1 200 mg/m³ air (analytical)
Remarks:
Insulin aspart, corresponding to 31.6 U/L (males and 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 of human Insulin male and female groups, respectively,
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

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to exposure to either of the test compounds.
One incident of convulsion occurred on Day 176, male animal 252 (receiving Human Insulin at 1979 mg/m3 suffered a slight convulsion that lasted for approximately 15 seconds at the pre-exposure examination. This animal had also suffered from reduced body tone and nervous behaviour earlier in the study (Days 130-142).
On Day 74, female satellite animal 398 (receiving Human Insulin at 1162 mg/m3) showed a marked unresponsive behaviour, underactivity, piloerection and hunched posture approximately ½ hour after dosing. Recovery was apparent approximately 2-3 min after the animal was given 2 mL of a solution of glucose (50% w/w) orally.

On Day 117, male animal 48 (receiving Insulin Aspart at 362 U/kg/day) also suffered a convulsion coupled with unresponsive behaviour.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four rats receiving 847 mg/m3 Human Insulin died during the first 8 weeks of the study and one later. There were no deaths at the higher doses. Two of the
early deaths were found dead in the restraint tube and are likely to have been procedural, the other two were found in the cage the next day prior to dosing but in the absence of deaths at higher doses, the relationship to treatment was considered to be unlikely.
Insulin Aspart treated rats showed a higher incidence of mortality at the high dose level for each sex. All high dose females died during the first 6 weeks of treatment, while the achieved dose in this group was 1796 mg/m3. This resulted in the decision to reduce the dose for this group from the middle of Week 6. At the lower dose of 1200 mg/m3 there was no further deaths during the remaining 19½ weeks of treatment. It was considered not necessary to reduce the dose for high dose males as there were only three deaths early in the study (plus one towards the end). The incidence of deaths at lower doses of Insulin Aspart was not clearly greater than that in controls.
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 for both test articles compared with concurrent controls.
There was no dose relationship.
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were considered to be no effects on haematological parameters attributable to treatment with either test substance.
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):
Mean bilirubin levels were statistically significantly increased in females receiving Insulin Aspart at 805 or 1200/1796 mg/m3 during Week 26 of treatment decreased in females receiving Human Insulin at 847 or 1162 mg/m3. Glucose concentrations (in samples collected in the morning approximately 22 hours after the last dose) were slightly increased in male groups receiving Human Insulin at 847 or 1979 mg/m3 during Weeks 13 and 26.

Comparatively, during Week 26, concurrent females receiving 847 or 1162 mg/m3 of Human Insulin showed a statistically significant decrease in glucose levels.
There was a general treatment related decrease in blood glucose levels in both male and female animals exposed to Insulin Aspart or Human Insulin on Day 1 and at Week 26.

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 treatment.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Higher urine volume was apparent in Weeks 13 and 26 for males receiving 2074 mg/m3 Insulin Aspart, in Week 26 only for males receiving 805 mg/m3 Insulin Aspart Week 26 only for males receiving 1979 mg/m3 Human Insulin.
There were no other changes considered to be treatment related effects for animals exposed to 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:
no effects observed
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 findingbeing a variable feature also in controls, this is considered to be background.
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
Human insulin
Effect level:
1 162 mg/m³ air (analytical)
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEC
Remarks:
Human insulin
Effect level:
1 979 mg/m³ air (analytical)
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEC
Remarks:
Insulin aspart
Effect level:
805 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
847 mg/m³ air (analytical)
System:
other: Blood suger regulation (Human Insulin)
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
156 mg/m³ air (analytical)
System:
other: Blood sugar regulation (Insulin aspart)
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for Human Insulin was 1162 mg/m3 in females and 1979 mg7m3 for males. Based on the hypoglycaemia associated deaths, the No Observed Adverse Effect Concentration (NOAEC) for Insulin Aspart was 805 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. Treatment related effects on bloodsugar were seen at all doses, as were expected due to the medical use of the product.
Executive summary:

Human Insulin and 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 Human insulin at:

Females:   0; 847; 1162 mg/m3

Males: 0; 847; 1979 mg/m3

And Insulin aspart at:

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 No Observed Adverse Effect Level (NOAEC) for Human Insulin was 1162 mg/m3 in females and 1979 mg7m3 for males.

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 Concnetration (NOAEC) for Insulin Aspart was 805 mg/m3.