Insulin DesB30

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data is available for the target substance.

In a study examining fertility and embryofetal development in rats dosed with 0, 100U/kg human insulin dosed twice daily by s.c. administration (corresponding to 0; 7.60 mg/kg/day) the males were dosed from 4 weeks before mating and females from 2 weeks before mating until day 15 of pregnancy. Reduced blood glucose levels were observed in relation to the dosing and one male rat died presumably due to severe hypoglycaemia. No effects on mating performance or pregnancy rate was observed, but slightly increased pre-and postimplanation losses was seen and a slight increase of foetuses with small orbital sockets was noted . Slightly reduced sperm count and slight histopathological changes in testes were found in males. The findings in the study was considered most likely to be induced by hypoglycaemia.

A study examining peri- and postnatal toxicity toxicity study was conducted with human insulin in female rats using dose levels of 0, 100U/kg dosed twice daily by s.c. administration (corresponding to 0; 7.60 mg/kg/day) from D6 of gestation to D20 after parturation. The administration of human insulin did not result in any effects on fertility or development. Hypoglycaemia was observed in the adult animals including a few lethal outcomes.

A developmental toxicity study in rabbits dosed with 0; 0.5; 1.5; 5 U/kg/ twice daily from day 6 to day 18 of pregnancy by s.c. injection (corresponding to 0; 0.04; 0.11; 0.38 mg/kg/d) resulted in an increase in early embryonic death and reduction of litter size at highest dose level and at all dose levels in a higher proportion of skeletal abnormalities. Blood glucose levels were affected in a dose-related manner and effects in relation to fertility and development were suggested to be secondary effects of lowered maternal blood glucose levels.

The findings from these studies are considered relevant for Insulin DesB30 as well as this substance also bind 100% to the insulin receptor and thus has the potential to induce a hypoglycaemic response. The data does not indicate any need for CLP-classification for fertility or development as the effects seen is primarily considered secondary to maternal toxicity. In addition, the dosing has been performed with s.c. administration, which make interpretation for more relevant exposure routes in terms of classification very difficult.

Link to relevant study records

Reference

Reference 1

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).

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.

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

Specific details on test material used for the study:

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

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

Dose / conc.:

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

Remarks:

Vehicle control group

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

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

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: 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

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)

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

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

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

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

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

No data is available for the target substance.

In a study examining fertility and embryofoetal development in rats dosed with 0, 100U/kg human insulin dosed twice daily by s.c. administration (corresponding to 0; 7.60 mg/kg/day) the males were dosed from 4 weeks before mating and females from 2 weeks before mating untill day 15 of pregnancy. Reduced blood glucose levels were observed in relation to the dosing and one male rat died presumably due to severe hypoglycaemia. No effects on mating performance or pregnancy rate was observed, but slightly increased pre-and postimplantation losses was seen and a slight increase of foetuses with small orbital sockets was noted . Slightly reduced sperm count and slight histopathological changes in testes were found in males. The findings in the study was considered most likely to be induced by hypoglycaemia.

A two-generation reproductive toxicity study was conducted with human insulin in rats using dose levels of 0, 100U/kg dosed twice daily by s.c. administration (corresponding to 0; 7.60 mg/kg/day). The administration of human insulin did not result in any effects on fertility or development. Hypoglycaemia was observed in the adult animals. Maternal toxicity was observed and was considered to be linked to the induction of hypoglycaemia and eight of 28 females died on day 20 to 23 of gestation.

A developmental toxicity study in rabbits dosed with 0; 0.5; 1.5; 5 U/kg/ twice daily from day 6 to day 18 of pregnancy by s.c. injection (corresponding to 0; 0.04; 0.11; 0.38 mg/kg/d) resulted in an increase in early embryonic death and reduction of litter size at highest dose level and at all dose levels in a higher proportion of skeletal abnormalities. Blood glucose levels were affected in a dose-related manner and effects in relation to fertility and development were suggested to be secondary effects of lowered maternal blood glucose levels.

The findings from these studies are considered relevant for Insulin DesB30 as well as this substance also bind 100% to the insulin receptor and thus has the potential to induce a hypoglycaemic response. The data does not indicate any need for CLP-classification for fertility or development as the effects seen is primarily considered secondary to maternal toxicity. Also, the dosing has been performed with s.c. administration which make interpretation for more relevant exposure routes in terms of classification very difficult.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

LOAEL

Effect level:

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)

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

Key result

Dose descriptor:

LOAEL

Effect level:

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)

other: (female only) pre and post implant losses

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

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

ophthalmological examination

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

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

Conclusions:

No data is available for the target substance. Data on fertility and development is only available on the active pharmaceutical ingredient (API) Human Insulin in connection with subcutaneous injections. This route of exposure is considered relevant since systemic effects are observed. The effects seen in relation to mortality in male rats is an effect of the pharmacological action of severe insulin-induced hypoglycemia. In addition the reduction in sperm count and motility and focal seminiferous epithelial atrophy with vacuolation of Sertoli cells in testes of male rats dosed 7.6 mg/kg/day is considered as a consequence of the hypoglycaemic mode of action of the substance and as secondary effects of the maternal toxicity. 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.

Insulin DesB30 is able to bind to the insulin receptor with equal affinity as Human Insulin, thus it is assumed to induce the same hypoglycemia-related effects.

Therefore, the effects seen from subcutaneous injection of Human Insulin is likely to occur in relation to oral, dermal or inhalational exposure to Insulin DesB30.

Executive summary:

Data on fertility and development is only available on the active pharmaceutical ingredient (API)  Human Insulin in connection with subcutaneous injections. This route of exposure is considered relevant since systemic effects are observed. The effects seen in relation to mortality in male rats is an effect of the pharmacological action of severe insulin-induced hypoglycemia. In addition the reduction in sperm count and motility and focal seminiferous epithelial atrophy with vacuolation of Sertoli cells in testes of male rats dosed 7.6 mg/kg/day is considered as a consequence of the hypoglycaemic mode of action of the substance and as secondary effects of the maternal toxicity. 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.

Insulin DesB30 is able to bind to the insulin receptor with equal affinity as Human Insulin, thus it is assumed to induce the same hypoglycemia-related effects.

Therefore, the effects seen from subcutaneous injection of Human Insulin is likely to occur in relation to oral, dermal or inhalational exposure to Insulin DesB30.

Effects on developmental toxicity

Description of key information

Data on fertility and development is only available on the active pharmaceutical ingredient insulin aspart in connection with subcutaneous injections. The effects seen in relation to maternal toxicity, fertility and development are overall considered as a consequence of the hypoglycaemic mode of action of the substance and as secondary effects of the maternal toxicity.

Justification for classification or non-classification

The findings from the the studies with human insulin are considered relevant for Insulin DesB30 as well as this substance also bind 100% to the insulin receptor and thus has the potential to induce a hypoglycaemic response. However, the data does not indicate any need for CLP-classification for fertility or development as the effects seen is primarily considered secondary to maternal toxicity. In addition, the dosing has been performed with s.c. administration, which make interpretation for more relevant exposure routes in terms of classification very difficult.

Based on weight of evidence Insulin DesB30 is not to be classified for reproductive toxicity according to the CLP-criteria.