1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Endpoint summary

Description of key information

Acute oral toxicity:

In an acute oral toxicity study in HanRcc: Wist(SPF) rats, following the acute toxic class method in accordance with the OECD guideline n° 423 and EU Method B.1 tris, the LD50 was determined to be 1000 mg/kg bw for female animals. The study has been performed in compliance with GLP (Van Sas P, 2017).

Acute inhalation toxicity:

No study is available. This endpoint is waived based on the following justification. A key study is available for the oral and dermal route of exposure. According to the REACH regulation, for substances other than gasses, only one additional route of exposure should be tested other than the oral route of exposure for acute toxicity (column 2, Annex VIII, section 8.5). Therefore, it is not necessary to perform an acute toxicity test via inhalation.

Acute dermal toxicity

In an acute dermal toxicity study in female wistar rats performed acording to the OECD guideline 402, the LD50 was established to exceed 2000 mg/kg bw (Van Sas P, 2019)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-09-08 to 2017-10-03

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)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147

Version / remarks:

November 2000; including the most recent partial revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16FD3167
- Expiration date of the lot/batch: 30 June 2018 (retest date)
- Purity test date: 98.2% (GC Assay)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: The preparations (w/w) were kept at room temperature and were dosed within 4 hours after adding the vehicle to the test item. Homogeneity was assessed by visual inspection of the solutions and the formulations were stirred during dosing, which ensures homogeneity sufficiently for these kinds of studies.
- Solubility and stability of the test substance in the solvent/vehicle: Stable for 6 hours. Adjustment was made for specific gravity of the vehicle. No correction was made for purity of the test item as the correction factor is 1.The concentration of the test item in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: Body weight variation did not exceed +/- 20% of the sex mean.
- Fasting period before study: Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test item. Water was available ad libitum.
- Housing: Group housing of 3 animals per cage in labeled Makrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 46 to 67%.
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

VEHICLE
- Concentration in vehicle: The concentration of the test item in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight: 200 mg/mL and 30 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: The vehicle was selected based on trial preparations performed at Charles River Den Bosch and on test item data supplied by the Sponsor. The vehicle was chosen from (in order of preference): water (Elix) (not tested based on instability in water), 1% aq. carboxymethyl cellulose (not tested based on instability in water), propylene glycol (spec.gravity 1.036) (cloudy solution), polyethylene glycol 400 (spec. gravity 1.125) (not tested according vehicle selection criteria) and corn oil (spec. gravity 0.92) (not tested according vehicle selection criteria). There was no information available regarding the solubility in vehicle.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg for all doses

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The first group was treated at a dose level of 2000 mg/kg. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines

Doses:

2000 mg/kg body weight; 300 mg/kg body weight

No. of animals per sex per dose:

3 females per dose group (2 groups)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Body weights: Days 1 (pre-administration), 8 and 15 and at death (if found dead after Day 1).
Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15.
The signs were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).
- Necropsy of survivors performed: The animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities recorded.
- Mortality/Viability: Twice daily.

Statistics:

No statistical analysis was performed

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

1 000 mg/kg bw

Based on:

test mat.

Mortality:

At 2000 mg/kg, two animals were found dead on Day 1 or 2.
At 300 mg/kg, no mortality occurred.

Clinical signs:

other: At 2000 mg/kg, uncoordinated movements, ptosis, piloerection and/or hunched posture were noted for the two animals found dead. The surviving animal showed lethargy, hunched posture, uncoordinated movements, piloerection, lean appearance and ptosis between

Gross pathology:

For one animal found dead, macroscopic post mortem examination revealed abnormalities of the stomach (greenish discoloration of the glandular mucosa), duodenum (gelatinous content), jejunum (gelatinous content) and ileum (gelatinous content). For the other animal found dead, macroscopic post mortem examination revealed abnormalities of the gastrointestinal tract (gelatinous content), trachea (gray white foamy content) and lungs (gray-white foamy content).
Macroscopic post mortem examination of the surviving animals did not reveal any abnormalities.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The oral LD50 value of JNJ-26144612-AAA (T002632) in Wistar rats was established to be within the range of 300-2000 mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 1000 mg/kg body weight.

Based on these results:
-according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), JNJ-26144612-AAA (T002632) should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route.
-according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), JNJ-26144612-AAA (T002632) should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

1 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-11-19 - 2019-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18IB2522
- Expiration date of the lot/batch: 2019-03-19 (retest date)
- Purity test date: 2018-09-20
- Appearance: White powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Stable for 6 hours in propylene glycol

OTHER SPECIFICS:
- Correction factor: 1

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approx. 10-12 weeks old)
- Weight at study initiation: 183 - 209 g; Body weight variation did not exceed +/- 20% of the mean.
- Fasting period before study: no data
- Housing: Individually housed in labeled Makrolon cages (MIII type, height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): ad libitum, free access to pelleted rodent diet
- Water (e.g. ad libitum): ad libitum, free access to tap water
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study. - Acclimation period: at least 5 days before start of treatment under laboratory conditions. During the acclimatization period the animals were group housed in Makrolon cages (MIV type, height 18 cm).
- Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the skin to be treated was intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 2018-11-19 To:2018-12-17

Type of coverage:

semiocclusive

Vehicle:

polyethylene glycol

Remarks:

specific gravity 1.036

Details on dermal exposure:

TEST SITE
- Area of exposure: the back of each animal
- % coverage: approx. 10% of the total body surface i.e. approx. 18 cm² for females
- Type of wrap if used: surgical gauze patch (Surgy 1D) successively covered with aluminum foil and Coban elastic bandage. A piece of Micropore tape was additionally used for fixation of the bandages.
- One day before exposure (Day -1) an area of approximately 5x7cm on the back of each animal was clipped.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 24 hours, after which dressings were removed and the skin cleaned of residual test item using tap water
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg (10 mL/kg) body weight
- Concentration: 200 mg/mL
- Constant volume or concentration used: yes

VEHICLE
- propylene glycol

PREPARATION OF FORMULATION
- The preparations (w/w) were kept at room temperature and dosed within 4 hours after adding the vehicle to the test item. Homogeneity was assessed by visual inspection of the solutions and the formulations were stirred during dosing, which ensures homogeneity sufficiently for these kinds of studies. Adjustment was made for specific gravity of the vehicle. No correction was made for purity of the test item as the correction factor is 1.
- Test item preparation was stirred on a magnetic stirrer during application.

Duration of exposure:

24 hours

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

3 females

Control animals:

not required

Details on study design:

- Duration of observation period following administration: until day 15 after treatment

Observations
- Mortality/viability: Twice daily
- Weighing: Days 1 (pre-administration), 8 and 15 and at death.
- Clinical signs: at periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15. The time of onset, degree and duration were recorded and the symptoms graded according to fixed scales:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).
- Necropsy: At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Descriptions of all internal macroscopic abnormalities were recorded.

Statistics:

No statistical analysis was performed.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No mortality occurred.

Clinical signs:

other: Chromodacryorrhoea of the snout was noted for two animals on Day 1 and hunched posture was noted for the other animal on Day 2. No further clinical signs were noted

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals

Other findings:

Irritation:
- no test item related erythema and edema was noted for the treated skin between Days 2 and 4
- Focal erythema, scales and scabs of the treated skin and scales and scabs of the right flank were seen in the animals during the observation period. The focal erythema was considered to be due to the dosing procedure rather than to be test item related, as test itel related erythema would show as general erythema of the treated skin area.

Interpretation of results:

GHS criteria not met

Conclusions:

The dermal LD50 value of JNJ-26144612-AAA (T002632) in Wistar Han rats was established to exceed 2000 mg/kg body weight.
Based on these results, JNJ-26144612-AAA (T002632) does not have to be classified and has no obligatory labelling requirement for acute dermal toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Acute oral Toxcity

An acute oral toxicity test was performed with T002632 according to the OECD guideline 423.

T002632 was administered by oral gavage to three female Wistar rats at 2000 mg/kg body weight. In a stepwise procedure two

additional groups of three females were dosed at 300 mg/kg body weight. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

At 2000 mg/kg, two animals were found dead on Day 1 or 2. At 300 mg/kg, no mortality occurred.

At 2000 mg/kg, uncoordinated movements, ptosis, piloerection and/or hunched posture were noted for the two animals found dead. The surviving animal showed lethargy, hunched posture, uncoordinated movements, piloerection, lean appearance

and ptosis between Days 1 and 13. At 300 mg/kg, piloerection and uncoordinated movements were noted for all animals on Day 1. Hunched posture was noted for three of the animals on Day 1.

The surviving animal at 2000 mg/kg showed reduced body weight gain during the first week and regained weight during the second week. At 300 mg/kg, the body weight gain shown by the animals over the study period was considered to be similar

to that expected for normal untreated animals of the same age and strain.

For one animal found dead, macroscopic post mortem examination revealed abnormalities of the stomach (greenish discoloration of the glandular mucosa),duodenum (gelatinous content), jejunum (gelatinous content) and ileum (gelatinous content). For the other animal found dead, macroscopic post mortem examination revealed abnormalities of the gastrointestinal tract (gelatinous content), trachea (gray white foamy content) and lungs (gray-white foamy content). Macroscopic post mortem examination of the surviving animals did not reveal any abnormalities.

The oral LD50 value of JNJ-26144612-AAA (T002632) in Wistar rats was established to be within the range of 300-2000 mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 1000 mg/kg body weight.

Acute inhalation toxicity

A key study is available for the oral and dermal route of exposure. According to the REACH regulation, for substances other than gases, only one additional route of exposure should be tested other than the oral route of exposure for acute toxicity (column 2, Annex VIII, section 8.5). Therefore, it is not necessary to perform an acute toxicity study via the inhalation route of exposure.

Acute dermal Toxicity

An acute dermal toxicity test was performed with T002632 according to the OECD guideline 402 (van Sas P, 2019). Initially, the test item was administered to a single female Wistar Han rat by a single dermal application at 2000 mg/kg body weight for24 hours in range finder study. Based on the results in the single female, the amin study was performed by dosing two females at 2000 mg/kg. All animals were subjected to daily observations and weekly determination of body weight.  Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

No mortality occurred. Chromodacryorrhoea of the snout was noted for two animals on Day 1 and hunched posture was noted for the other animal on Day 2. No further clinical signs were noted. No test item related erythema and edema was noted for the treated skin between Days 2 and 4. The changes noted in body weight gain were within the range expected for rats used in this type of study and were therefore considered not indicative of toxicity.

No abnormalities were found at macroscopic post mortem examination of the animals.

The dermal LD50 value of JNJ-26144612-AAA (T002632) in Wistar Han rats was established to exceed 2000 mg/kg body weight.

Justification for classification or non-classification

Based on the results on oral LD50 ranging between 300 - 2000 mg/kg/bw and according to the criteria laid down in the EC regulation No 1272/2008 on classification, labelling and packaging of substances and mixtures, T002632 should be classified as category 4 and should be labeled as H302: harmful if swallowed.

No data were available to decide on the classification for the inhalation route.

Based on the dermal LD50 exceeding 2000 mg/kg bw, T002632 does not have to be classified and has no obligatory labelling requirement for acute dermal toxicity according to the criteria laid down in Regulation (EC) No 1272/2008.