Insulin Human Methyl Ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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-20, study nr. 940303, 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.

The study is a fertility and Embryofoetal developmental toxicity study in the Rat performed in accordance with ICH test guidelines 4.1.1 + 4.1.3 (comparable to OECD 421/422).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Subcutaneous administration used instead of oral administration.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Subcutaneous administration used instead of oral administration.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

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:

Study performed with the active pharmaceutical ingredient Human Insulin (Actrapid)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

not specified

Details on exposure:

NA

Details on mating procedure:

NA

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Female rats were treated for 2 weeks prior mating and dosing continued up to and including day 15 of pregnancy (total of 29 days).
Male rats were treated for 4 weeks prior mating and dosing continued up to day 20 of pregnancy (total of 48 days).

Frequency of treatment:

Twice daily. S.C administration.

Details on study schedule:

NA

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control group

Doses / concentrations 2

Dose / conc.:

7.6 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a previous study
the daily dose level was distrubuted into subcutaneous injections twice daily

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: NA

BODY WEIGHT: Yes
- Time schedule for examinations: NA

-Other:
- Food and water consumption was recorded, although not a feeding or water study.

Sperm parameters (parental animals):

Parameters examined in [all] male rats:
[testis weight, sperm count, sperm motility, sperm morphology]

Litter observations:

STANDARDISATION OF LITTERS
- not specified

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and distrubution, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain].
Live young were examined externally and weighted. Half the fetuses were examined for viceral abnormalities and half were examined for skeletal abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
[ yes, for external and internal abnormalities]

Postmortem examinations (parental animals):

SACRIFICE
- Female and Male animals killed at Day 20 of pregnancy.

GROSS NECROPSY
- Yes, not specified

HISTOPATHOLOGY / ORGAN WEIGHTS
- Congenital abnormalities and number of corpora lutea (maternal animals)
- male reproductive system including, testes and sperm (parental animals)

Postmortem examinations (offspring):

SACRIFICE

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera, Skeletal morphology and Orbital socklet morphology.]


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

Death of one male animal 7.60 mg/kg/day. This effect was most likely related to the pharmacological action of human insulin (Actrapid) causing severe hypoglycemia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Weight gain (and increased food and water intake) observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dosage related increase in food and water intake was seen, although the study is not a feeding study.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma glucose was reduced from about 10 mmol/l to 4-6 mmol/l at one hour after dosing with 7.60 mg/kg/day, rate of recovery showing some dosage-dependency.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Slightly reduced pre- and post implantation losses were observed at 7.60 mg/kg/day.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Slightly reduced sperm counts and motility and focal seminiferous epithelial atrophy with vacuolation of Sertoli cells in testes at 7.60 mg/kg/day.

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 7.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

7.6 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Detailed foetal examination revealed a slight dosage related increase in the incidence of fetuses with absent or small orbital sockets at 7.60 mg/kg/day.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

The only foetal effects observed was absent or small orbital sockets at all doses. No other abnormalities reported.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

ca. 7.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

ophthalmological examination

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

7.6 mg/kg bw/day (actual dose received)

System:

other:

Organ:

other: ophthalmological abnormalities

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

7.6 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

A fertility and embryofoetal developmental toxicity study in the Rat performed in accordance with ICH test guidelines 4.1.1 + 4.1.3 (comparable to OECD 421/422). 24 male and 24 female rats were daily dosed subcutaneously with Human Insulin (0, 7.6 mg/kg/ bw/day) for 4-7 weeks. Mortality was observed in one male at highest dose level, an effect of the pharmacological action of severe insulin-induced hypoglycemia. However, terminal studies on male rats also revealed a slightly reduced sperm count and and motility and histopathology showed focal seminiferous epithelial atrophy with vacuolation of Sertoli cells in testes of male rats dosed 7.6 mg/kg/day. Detailed foetal examination revealed a slight dosage related increase in fetuses with absent or small orbital socket. No other toxicity was observed in parental or foetal animals.

Executive summary:

A fertility and embryofoetal developmental toxicity study in the Rat performed in accordance with ICH test guidelines 4.1.1 + 4.1.3 (comparable to OECD 421/422). 24 male and 24 female rats were daily dosed subcutaneously with Human Insulin (0, 7.6 mg/kg/ bw/day) for 4-7 weeks. Mortality was observed in one male at highest dose level, an effect of the pharmacological action of severe insulin-induced hypoglycemia. However, terminal studies on male rats also revealed a slightly reduced sperm count and and motility and histopathology showed focal seminiferous epithelial atrophy with vacuolation of Sertoli cells in testes of male rats dosed 7.6 mg/kg/day. Detailed foetal examination revealed a slight dosage related increase in fetuses with absent or small orbital socket. No other toxicity was observed in parental or foetal animals.