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Administrative data

Description of key information

Single dose oral toxicity studies in mice and rats revealed NOELs of 2000 mg/kg in both species. In single dose intraperitoneal studies in mice and rats, a NOEL of 300 mg/kg was reported in mice but a NOEL was not identified in rats.

The inhalatory LC50, 4h value of the substance in Wistar rats was established to exceed 5 mg/L.

The dermal LD50 value of the substance in Wistar rats was established to exceed 2000 mg/kg body weight.

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:
Nov 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Single dose acute toxicity testing for pharmaceuticals: Revised guideline. Federal Register, August 26. 1998. p 43934-43935
Version / remarks:
Chengelis 1995
GLP compliance:
yes
Test type:
acute toxic class method
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
3 males and 3 females/ group ( Groups 1 - 4)
5 males and 5 females/group (Group 5)


Room temperature: 18° to 26°C
Room relative humidity: 30 to 70%
Lighting cycle: Artificial light for a 12-hour light/12-hour dark cycle
Animal caging: In stainless steel wire cages in compliance with the NRC
(National Research Council) Guide for the Care and Use of Laboratory Animals.
Diet: Teklad Certified LM-485 Rodent Diet #7012C ad libitum. except approximately 4 hours prior to dose (fast) until approximately 2 hours post-dose.
Analysis of diet: The lot number(s) and specifications of each lot used are archived at Calvert.
Water was provided to the animals ad libitum
Periodic analyses of the water were performed and the results are archived at Calvert.

Animals were housed individually and in compliance with the National Research Council
"Guide for the Care and Use of Laboratory Animals". Temperature and relative humidity
were monitored daily. Calvert is USDA registered and a fully accredited AAALAC facility.

There were no known contaminants in the diet or water which, at the levels detected.
interfered with the purpose, conduct or outcome of the study.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) sodium carboxymethylcellulose [CMC] (high viscosity 1500-3000 cps)
Details on oral exposure:
Route and frequency: Oral, once to each rat
Dose volume: 20 mL/kg
AI-IU377 in 0.5% (w/v) sodium carboxymethylcellulose (0.5% CMC). The actual dose preparation procedures were documented in the raw data and presented as follows:
The amount of AHU3 77 required for each batch was based on the batch size.
Dosing formulations were prepared by gradual addition of 0.5% CMC to the calculated amount of test article, using a mortar and pestle. The suspensions were transferred to an appropriate HPDE container and the mortar and pestle were rinsed with vehicle. A stir bar was added and the mixture was thoroughly mixed for over an hour. The contents were then transferred to a graduated cylinder and the remaining vehicle was added. The formulations were then transferred
back into the HOPE container. The following preparations reflect the total amounts used to make each final dosing formulation.
Doses:
Five groups consisting of five animals per sex per group were allocated to the study. Four groups consisting of three animals per sex per group were dosed at 0. 50. 250 and 1000 mg/kg on Day I. Since neither lethality nor other relevant clinical signs were observed. the remaining two animals per sex of Groups 1 thru 4 were not dosed and all animals allocated to Group 5 (5/sex) were dosed at 2000 mg/kg.
No. of animals per sex per dose:
Five groups consisting of five animals per sex per group were allocated to the study. Four groups consisting of three animals per sex per group were dosed at 0. 50. 250 and 1000 mg/kg on Day I. Since neither lethality nor other relevant clinical signs were observed. the remaining two animals per sex of Groups 1 thru 4 were not dosed and all animals allocated to Group 5 (5/sex) were dosed at 2000 mg/kg.
Control animals:
yes
Details on study design:
Pretest period: A minimum of at least 5 days was allowed between animal receipt and the start of dosing to acclimate animals to the laboratory environment and to observe them for the development of infectious disease.
Study period: Fifteen days
The test article dosing preparations were administered once to each rat orally. Each animal received 20 mL/kg via a syringe (5 cc) and a 16 g gavage needle. The dosing formulations were shaken then stirred via a magnetic stir plate and stir bar prior to dosing and continued during dosing. Unused portions of dosing formulations were discarded at the end of each dosing day.
Key result
Sex:
male/female
Dose descriptor:
other: no-effect level
Effect level:
2 000 mg/kg bw
Based on:
test mat.
Mortality:
There was no mortality
Clinical signs:
No clinical signs were observed in any animals receiving the vehicle formulation or the test
article at 50, 250, 1000 and 2000 mg/kg during the study with the exception of soft feces in
one animal at the 6 hour observation at the 2000 mg/kg dose level.
Body weight:
Body weights were recorded as per protocol. No biologically significant differences were
observed.
Gross pathology:
There were no gross necropsy findings in any of the animals receiving the vehicle formulation
or the test article at 50, 250, 1000 or 2000 mg/kg.
Interpretation of results:
other: no-effect level was identified at 2000 mg/kg.
Conclusions:
Single oral administration of AHU377 at 50, 250, 1000 and 2000 mg/kg to rats revealed no clinical signs with an absence of mortality. Under the conditions of this study, a no-effect level was identified at 2000 mg/kg.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Qualifier:
according to
Guideline:
other: JMAFF Guidelines (2000)
GLP compliance:
yes
Test type:
acute toxic class method
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
5 males and 5 females (females were nulliparous and nonpregnant)
Animals used within the study were of approximately the same age and body weight variation did not exceed +/- 20% of the sex mean.
Identification Individual unique number by tattoo on hind leg.
Health inspection At least prior to exposure. It was ensured that the animals were healthy and without any abnormality that might affect the study integrity.
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 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.
Accommodation
Group housing of five animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm) containing sterilised 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).
Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany) except during exposure to the test substance.
Water
Free access to tap water except during exposure to the test substance.
Animal husbandry on the day of exposure
The animals were moved to the inhalation area to in order to perform the exposure. During the exposure, there was no access to food and water. After exposure, the animals were returned to their cages which were placed in a fume cupboard for a short time period to allow test
substance remnants to evaporate. A sheet of filter paper was used to cover the bedding material to prevent suffocation in case of bad health condition and in order to recover and to aid the clinical observations. The sheet was removed and before the end of the exposure day, the
surviving animals were returned to the animal room.
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.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.5 - <= 3.7 µm
Geometric standard deviation (GSD):
>= 1.9 - <= 2.4
Remark on MMAD/GSD:
The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the exposure period. The MMAD was 3.5 μm (gsd 1.9) and 3.7 μm (gsd 2.4)
Details on inhalation exposure:
Administering the test substance to a stream of pressurized air using a combination of a spiral feeder and micronizing jet mill generated an aerosol. The aerosol was passed through a series of three cyclones, allowing larger particles to settle, before it entered the exposure chamber. The mean airflow 11 L/min.
From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
The nominal concentration was calculated by dividing the amount of test substance used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the
equilibrium time was negligible. The volume of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the exposure time.
The actual concentration was determined eighteen times during the exposure period. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. Samples were drawn through a glass fiber filter
(type APFC04700, Millipore, Billerica, MA, USA). The collected amount of the test substance in the air sample was measured gravimetrically. Sample volumes were measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands).
Subsequently the time-weighted mean concentration with the standard deviation was calculated.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
4 hours to a target
concentration of the test substance of 5 mg/L
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
Target concentrations were based on the cut off concentration values specified in the UN and
EC classification guidelines. Five animals of each sex were exposed for 4 hours to a target
concentration of the test substance of 5 mg/L.
Prior to exposure the animals were restrained in polycarbonate restraining tubes; these tubes
were connected to the exposure chamber. Twenty-three minutes after the last animal was
placed the generation of the test atmosphere was started. The exposure time was 4 hours.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
Three males were found dead. Two males were found dead 18 minutes after the start of the
exposure and the third male was found dead 4 minutes after completion of the exposure. No
further mortality occurred.
Clinical signs:
other: During exposure, laboured and shallow respiration was seen for all animals observed (not presented in the table). After exposure, lethargy, hunched posture, laboured respiration, piloerection, ptosis was seen for the animals. The surviving animals had rec
Body weight:
Body weight gain in one surviving male and all females was within the range expected for rats of
this strain and age used in this type of study. One male showed body weight loss up to Day 8
and regained body weight during the second week post-exposure.
Gross pathology:
Macroscopic post mortem examination of the three males that were found dead during the study
revealed an enlarged heart in one animal, foamy contents of the trachea in another animal and
granulated gray-white contents of the trachea in all three animals. No abnormalities were noted
for the surviving animals.
Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The inhalatory LC50, 4h value of Calcium bis (4-{[ (1S, 3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate) in Wistar rats was established to exceed 5 mg/L.
Based on these results and according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS), United Nations, New York and Geneva (2011) (including all amendments), Calcium bis (4-{[ (1S, 3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate) should be classified in Category 5 for acute toxicity by the inhalation route.
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), Calcium bis (4-{[ (1S, 3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate) does not have to be classified and has no obligatory labelling requirement for inhalation toxicity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
5 000 mg/m³

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:
Oct 2014 - Apr 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Qualifier:
according to
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Qualifier:
according to
Guideline:
other: JMAFF Guidelines (2000)
GLP compliance:
yes
Test type:
fixed dose procedure
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Rat, Wistar strain, Crl:WI (Han) (outbred, SPF-Quality).
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 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.
Accommodation
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).
Acclimatization period was 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).
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water
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.
Type of coverage:
not specified
Vehicle:
water
Details on dermal exposure:
One day before exposure (Day -1) an area of approximately 5x7 cm on the back of each animal was clipped.
The formulation was applied on an area of approx. 10% of the total body surface, i.e. approx. 25 cm² for males and 18 cm² for
females. The formulation was held in contact with the skin with a dressing, consisting of a 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 in females only.
Duration of exposure:
Single dosage, on Day 1
Doses:
2000 mg/kg (10 mL/kg) body weight
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
Calcium bis (4-{[ (1S, 3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate) was administered to five Wistar rats of each sex by a single dermal application at 2000 mg/kg body weight for 24 hours. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice
(Day 15).
The study was carried out based on the guidelines described in:
OECD No.402 (1987) "Acute Dermal Toxicity"
Commission Regulation (EC) No 440/2008, B3: "Acute Toxicity (Dermal)"
EPA, OPPTS 870.1200 (1998), "Acute Dermal Toxicity"
JMAFF Guidelines (2000), including the most recent revisions.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred.
Clinical signs:
No clinical signs were noted for the animals.
White staining was seen on the treated skin-area of all animals on Days 2 and/or 3. Additionally,
scales were noted for one animal between Days 4 and 7.
Body weight:
The changes noted in body weight gain in males and females were within the range expected for rats used in this type of study and were therefore considered not indicative of toxicity.
Gross pathology:
No abnormalities were found at macroscopic post mortem examination of the animals.
Interpretation of results:
GHS criteria not met
Conclusions:
The dermal LD50 value of Calcium bis (4-{[ (1S, 3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate) in Wistar rats was established to exceed 2000 mg/kg body weight.
Based on these results, Calcium bis (4-{[ (1S, 3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoate) 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 (2011) (including all amendments).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

Additional information

Justification for classification or non-classification

Single dose oral toxicity studies in mice and rats revealed NOELs of 2000 mg/kg in both species. In single dose intraperitoneal studies in mice and rats, a NOEL of 300 mg/kg was reported in mice but a NOEL was not identified in rats.

The inhalatory LC50, 4h value of AHU 377 in Wistar rats was established to exceed 5 mg/L.

Based on these results and according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS), United Nations, New York and Geneva (2011) (including all amendments), AHU 377 should be classified in Category 5 for acute toxicity by the inhalation route.

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), AHU 377 does not have to be classified and has no obligatory labelling requirement for inhalation toxicity.

The dermal LD50 value of AHU 377 in Wistar rats was established to exceed 2000 mg/kg body weight.

Based on these results, AHU 377 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 (2011) (including all amendments).