Insulin Aspart Precursor

Endpoint summary

Description of key information

Acute oral toxicity studies (OECD 423) on the structural analogues MI3 and X14DesB30 both resulted in LD50 valus above 2000 mg/kg bw, as no lethaliiy occured at the highest dose levels of 2000 mg/kg bw. Thus, due to close structural similarity to these substances  low acute oral toxicity (LD50 > 2000 mg/kg) can be concluded for  Insulin aspart precusor as well. The  low potential for acute oral toxicity is further supported by the data indicating very low -if any- oral absorption of the substance see section 7.1.1.

Data obtained from an OECD 402 study on MI3 indicate very low acute dermal toxicity potential as LD50 > 2000 mg/kg bw. Thus, due to close structural similarity to MI3 an dermal LD50 > 2000 mg/kg bw can be concluded for Insulin aspart precusor as well. The absence of dermal acute toxicity is further supported by the lack of dermal uptake of the subtance as indicated in 7.1.1.

For further read-across justification see document attached in section 13.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute 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

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Interpretation of results:

GHS criteria not met

Conclusions:

Read-across from acute oral toxicity studies on two closely structural and physicochemical related substances: MI3 (that cannot bind to the insulin receptor) and X14DesB30 (that bind to the insulin receptor). For both substances no lethality was noted at the highest test dose level of 2000 mg/kg/bw.
Thus very low potential for acute toxicity by the oral route can be expected for insulin aspart precursor as well and thus, the criteria for CLP-classification for acute oral toxicity is not met.
For further read-across justification se document attached in section 13.

Executive summary:

Read-across from acute oral toxicity studies on two closely structural and physicochemical related substances: MI3 (that cannot bind to the insulin receptor) and X14DesB30 (that bind to the insulin receptor). For both substances no lethality was noted at the highest test dose level of 2000 mg/kg/bw.

Thus very low potential for acute toxicity by the oral route can be expected for insulin aspart precursor as well and thus, the criteria for CLP-classification for acute oral toxicity is not met.

For further read-across justification se document attached in section 13.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.03.03 -2000.07.04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

Storage temperature was < -10 oC. The animals were transferred from another study and had a longer acclimatization period so they approximated to the protocol age range. The changes had no influences on the integrity or the results of the present study

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

Storage temperature was < -10 oC. The animals were transferred from another study and had a longer acclimatization period so they approximated to the protocol age range. The changes had no influences on the integrity or the results of the present study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

Storage temperature was < -10 oC. The animals were transferred from another study and had a longer acclimatization period so they approximated to the protocol age range. The changes had no influences on the integrity or the results of the present study

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (MI3 dried)
- Source and lot/batch No.of test material: C202483
- Expiration date of the lot/batch: 2001.05.31
- Purity test date: 2001.05.31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -10 degrees Celcius
- Stability under test conditions: NA
- Solubility and stability of the test substance in the solvent/vehicle: Full solubility in vehicle (water)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Purified water (Elgastat purifier used)
- Final preparation of a solid: 100mg/ml MI3 in water

FORM AS APPLIED IN THE TEST (if different from that of starting material): solid disolved in purified water.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:WI(Glx/BRL/Han)BR)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 183 -198g (males); 130 - 149g (females)
- Fasting period before study: yes, overnight fasting.
- Housing: Three rats of same sex per cage (suspended stainless steel mesh cage).
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 22- 24 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 degrees Celcuis.
- Humidity (%): 40 - 70%.
- Air changes (per hr): a minimum of 14 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12- / 12 hours

IN-LIFE DATES: From: To: 36 - 38 days.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 100 mg/ml
- Amount of vehicle (if gavage): 20 ml/kg bw.
- Justification for choice of vehicle: MI3 was dispersed in purified water, which is non-toxic in the volume given.
- Lot/batch no. (if required): C202483
- Purity: Pure water (Elgastat purifier)

MAXIMUM DOSE VOLUME APPLIED: 20 ml/kg bw.

DOSAGE PREPARATION (if unusual): NA

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Dose levels were chosen in complience with limits stated in the guidelines - i.e. first dose 2000 mg/kg to three animals of one sex.

Doses:

2000 mg/kg bw.

No. of animals per sex per dose:

3 (females) / 3 (males)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Clinical signs:The day of dosing (fist day), one within half an hour of dosing and four times within the first four hours after administration. Twice daily on Days 2, 3 and 4, and once daily from the fifth to the last day of the observation period. Weighing: one day before dosing (day -1), day 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,

Statistics:

None

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat. (total fraction)

Mortality:

There were no deaths following a single oral dose of MI3 of both sexes dosed at 2000 mg/kg.

Clinical signs:

There were no clinical signs of reaction to treatment were apparent.

Body weight:

All rats gained weight during the first and second week of the observation period.

Gross pathology:

No macroscopic changes were observed for animals killed on Day 15.

Other findings:

NA

Interpretation of results:

GHS criteria not met

Remarks:

oral LD50 >2000 mg/kg bw.

Conclusions:

The study was conducted to access the acute oral toxicity of MI3 given as single dose to a small group of rats of both sex. The study was designed in accordance with OECD guideline method 423. Groups of three male or female rats were fasted overnight and given a single dose of MI3 oral gavage at a dose level of 2000 mg/kg. All animals were killed at day 15 after dosing. No death, clinical signs or post mortem abnormalities were observed for rats of both sexes after a single oral dose of 2000 mg/kg. The median LD50 of MI3 in rats was found to be > 2000 mg/kg bw.

Executive summary:

The Study was conducted to access the acute oral toxicity of MI3 given as single dose to a small group of rats of both sex. The study was designed in accordance with OECD guideline method 423, the commission directive 94/54/EEC, Method B1 tris, and the US EPA Health effects test Guidelines OPPTS 870.1100.

The study design provided information for hazard assessment and classification and enables the MI3 to be assigned to one of the four toxicity classes identified in Annex of Commission Directive 93/21/EEC, while severely restricting animal usage.

Groups of three male or female rats were fasted overnight and given a single dose of MI3 oral gavage at a dose level of 2000 mg/kg. The MI3 was dispersed in purified water and dosed at a dose volume of 20 mL/kg. All animals were killed at day 15 after dosing and underwent a full necropsy.

No deaths were observed for rats of both sexes after a single oral dose of 2000 mg/kg. No clinical signs were observed, and all rats (both sexes) gained weight the two weeks following dosing. Necropsy did not reveal any macroscopic abnormalities among the dosed animals.

The median LD50 of MI3 in rats was found to be > 2000 mg/kg bw.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.03.31 - 2000.07.04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (X14DesB30, solid)
- Source and lot/batch No.of test material: X1YIS0201014
- Expiration date of the lot/batch: NA
- Purity test date: 2000.02.18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -70 degrees Celcius
- Stability under test conditions: NA
- Solubility and stability of the test substance in the solvent/vehicle: Full solubility in vehicle (water)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Purified water (Elgastat purifier used)
- Final preparation of a solid: 100mg/ml X14DesB30 in water

FORM AS APPLIED IN THE TEST (if different from that of starting material): solid disolved in purified water.

Species:

rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:WI(Glx/BRL/Han)BR)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 278 – 336g (males); 180 - 194g (females)
- Fasting period before study: yes, overnight fasting.
- Housing: Three rats of same sex per cage (suspended stainless steel mesh cage).
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 14 - 16 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 degrees Celcuis.
- Humidity (%): 40 - 70%.
- Air changes (per hr): a minimum of 14 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12- / 12 hours

IN-LIFE DATES: From: To: 29 - 31 days.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 100 mg/ml
- Amount of vehicle (if gavage): 20 ml/kg
- Justification for choice of vehicle: MI3 was dispersed in purified water, which is non-toxic in the volume given.
- Lot/batch no. (if required): C202483
- Purity: Pure water (Elgastat purifier)

MAXIMUM DOSE VOLUME APPLIED: 20 ml/kg bw

DOSAGE PREPARATION (if unusual): NA

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Dose levels were chosen in complience with limits stated in the guidelines - i.e. first dose 2000 mg/kg to three animals of one sex.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 (females) / 3 (males)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Clinical signs: The day of dosing (fist day), one within half an hour of dosing and four times within the first four hours after administration. Twice daily on Days 2, 3 and 4, and once daily from the fifth to the last day of the observation period. Weighing: one day before dosing (day -1), day 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,

Statistics:

NA

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat. (total fraction)

Mortality:

There were no deaths following a single oral dose of X14desB30 of both sexes dosed at 2000 mg/kg.

Clinical signs:

There were no clinical signs of reaction to treatment were apparent apart from a single transient observation of pilo-erection for one female one hour after dosing.

Body weight:

Two male and three females showed low weight gains or small losses between Day -1 and Day 8. The females continued to show small gains during the second week of the study.
The majority of rats gained weight during the second week of their observation period.

Gross pathology:

No macroscopic changes were observed for animals killed on Day 15.

Other findings:

NA

Interpretation of results:

GHS criteria not met

Conclusions:

The Study was conducted to access the acute oral toxicity of X14DesB30 given as single dose to a small group of rats of both sex. The study was designed in accordance with OECD guideline method 423. Groups of three male or female rats were fasted overnight and given a single dose of X14DesB30 by oral gavage at a dose level of 2000 mg/kg. All animals were killed at day 15 after dosing. No death or post mortem abnormalities were observed for rats of both sexes after a single oral dose of 2000 mg/kg. However, a single transient observation of pilo-erection was observed for one female one hour after dosing. The median lethal dose 50 of X14DesB30 in rats was found to be > 2000 mg/kg bw.

Executive summary:

The Study was conducted to access the acute oral toxicity of X14DesB30 given as single dose to a small group of rats of both sex. The study was designed in accordance with OECD guideline method 423, the commission directive 94/54/EEC, Method B1 tris, and the US EPA Health effects test Guidelines OPPTS 870.1100.

The study design provided information for hazard assessment and classification and enables the X14DesB30 to be assigned to one of the four toxicity classes identified in Annex of Commission Directive 93/21/EEC, while severely restricting animal usage.

Groups of three male and female rats were fasted overnight and given a single dose of MI3 oral gavage at a dose level of 2000 mg/kg. The X14DesB30 was dispersed in purified water and dosed at a dose volume of 20 mL/kg. All animals were killed at day 15 after dosing and underwent a full necropsy.

No deaths were observed for rats of both sexes after a single oral dose of 2000 mg/kg. No clinical signs were observed, apart from a single transient observation of pilo-erection for one female one hour after dosing. Two male and three females showed low weight gains or small losses the first week. The females continued to show small gains during the second week of the study, although the majority of rats gained weight during the second week of their observation period. Necropsy did not reveal any macroscopic abnormalities among the dosed animals.

The median lethal dose 50 of X14DesB30 in rats was found to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: dermal

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

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 000 mg/kg bw

Based on:

test mat. (total fraction)

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Interpretation of results:

GHS criteria not met

Conclusions:

No data is available for Insulin aspart precurser (T). Data obtained from an OECD 402 study on MI3 (S2) indicate very low acute dermal toxicity potential as LD50 > 2000 mg/kg bw. Thus, due to close structural similarity to MI3 an dermal LD50 > 2000 mg/kg bw can be concluded for Insulin aspart precusor as well. The absence of dermal acute toxicity is furhter supported by the lack of dermal uptake of the subtance as indicated in 7.1.1.
The criteria for CLP classification for acute dermal toxicity of insulin aspart precursor is not met.

Executive summary:

No data is available for Insulin aspart precurser (T). Data obtained from an OECD 402 study on MI3 indicate very low acute dermal toxicity potential as LD50 > 2000 mg/kg bw. Thus, due to close structural similarity to MI3 an dermal LD50 > 2000 mg/kg bw can be concluded for Insulin aspart precusor as well. The absence of dermal acute toxicity is furhter supported by the lack of dermal uptake of the subtance as indicated in 7.1.1.

The criteria for CLP classification for acute dermal toxicity of insulin aspart precursor is not met.

Reference 2

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.03.03 - 2000.07.04.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

Storage temperature was < -10 oC. The animals were transferred from another study and had a longer acclimatization period so they approximated to the protocol age range. The changes had no influences on the integrity or the results of the present study

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

yes

Remarks:

Storage temperature was < -10 oC. The animals were transferred from another study and had a longer acclimatization period so they approximated to the protocol age range. The changes had no influences on the integrity or the results of the present study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

Storage temperature was < -10 oC. The animals were transferred from another study and had a longer acclimatization period so they approximated to the protocol age range. The changes had no influences on the integrity or the results of the present study

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL (MI3 dried)
- Source and lot/batch No.of test material: C202483
- Expiration date of the lot/batch: 2001.05.31
- Purity test date: 2001.05.31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -10 degrees Celcius
- Stability under test conditions: NA
- Final preparation of a solid: dosed as powder/soild at 2000mg/kg bw

FORM AS APPLIED IN THE TEST (if different from that of starting material): as solid/ powder

Species:

rat

Strain:

Wistar

Remarks:

CRL:Han Wist(Glx/BRL)BL)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:WI(Glx/BRL/Han)BR)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 188 -208g (males); 137 - 163g (females)
- Fasting period before study: yes, overnight fasting.
- Housing: Three rats of same sex per cage (suspended stainless steel mesh cage).
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 22- 24 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 degrees Celcuis.
- Humidity (%): 30 - 70%.
- Air changes (per hr): a minimum of 14 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12- / 12 hours

IN-LIFE DATES: 31- 38 days.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

Wetting of the application area with 0.2 mL water before adding MI3 powder

Details on dermal exposure:

TEST SITE
- Area of exposure: 5 x 5 cm
- % coverage: 10% of the total body surface
- Type of wrap if used: Semi occlusive

REMOVAL OF TEST SUBSTANCE
- Washing (if done): brushed and swabbed with moist cotton wool
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): NA
- Constant volume or concentration used: NA
- For solids, paste formed: no

VEHICLE
Not used, application area moistened with water before application of powder.

Duration of exposure:

24 hours.

Doses:

single dose of 2000 mg/kg bw.

No. of animals per sex per dose:

2 female rats (Preliminary study) and 5 males and 5 females (main study)

Control animals:

no

Details on study design:

Duration of observation period following administration: 8 days (preliminary study) 14 days (main study)
- Frequency of observations and weighing:
Clinical signs:The day of dosing (fist day), once within half an hour of dosing and four times within the first four hours after administration. Twice daily on Days 2, 3 and 4, and once daily from the fifth to the last day of the observation period. Weighing: one day before dosing (day -1), day 1, 8 and 15.
Dermal reactions were recorded from day 2 of exposure and similar to that for clinical signs.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,

Statistics:

NA

Preliminary study:

Yes: Two female rats dosed at 2000 mg/kg bw. Based on the results these investigations the limie dose level was selected for the main study. Since no compound-related motality occured in the preliminary test, a group of five male and female rats was subjected to a single dermal application of MI3 at 2000 mg/kg bw.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat. (total fraction)

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

No mortality was observed neither in the preliminary study nor the main study.

Clinical signs:

- No signs in the preliminary study.
- Three incidence of anogenital soiling for 3 female rats at day 2, recovery at day 3. No dermal reactions both sex.

Body weight:

all rats gained weight during the first and second week.

Gross pathology:

One male and female rat with slight pelvic dilatation of one kidney.

Other findings:

NA

Interpretation of results:

GHS criteria not met

Conclusions:

The Study was conducted to access the acute dermal toxicity of MI3 given as single dose to a small group of rats of both sex. The study was designed in accordance with OECD guideline method 402. In a preliminary study two female rats and in the main study groups of five male and female rats were given a single dose of MI3 at a dose level of 2000 mg/kg bw. All animals were killed at day 8 (preliminary study) or day 15 (main study) after dosing. No death were observed, in the main study transient anogenital soiling was observed among three female rats, and one male and one female rat had slight pelvic dilatation of one kidney. The median LD50 of MI3 in rats was found to be > 2000 mg/kg bw.

Executive summary:

The Study was conducted to access the acute dermal toxicity of MI3 given as single dose to a small group of rats of both sex. The study was designed in accordance with OECD guideline method 402, the commission directive 92/69/EEC, Method B3, and the US EPA Health effects test Guidelines OPPTS 870.1200.

In the preliminary study two female rats had undiluted MI3 applied to the dorsa at a dose level of 2000 mg/kg bw. In the main study, groups of five male and female rats were given a single topical application of MI3 at a dose level of 2000 mg/kg bw to the dorsum with a semi occlusive bandage covering the dose site for 24 hours. Animals of the preliminary study were killed at day 8, while animals of the main study were killed at day 15. All animals underwent a full necropsy.

Weight gain was recorded among all animals during the study period. In the preliminary study, no death or macroscopic changes were evident 8 days after dosing. In the main study, transient anogenital soiling was observed among three female rats on day 2 with recovery on day 3. On day 15 one male and one female rat had slight pelvic dilatation of one kidney.

The median LD50 of MI3 in rats was found to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

mg/kg bw

Justification for classification or non-classification

Low acute ptoxic potential is concluded for Insulin aspart precursor as oral and dermal LD50 values were concluded to be above 2000 mg/kg bw. Thus, Insulin aspart precusor is not to be classified for acute oral - or dermal - toxicity according to the CLP-classification criteria.