Registration Dossier

Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Sep 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 403 (Acute Inhalation Toxicity)
according to
EU Method B.2 (Acute Toxicity (Inhalation))
according to
EPA OPPTS 870.1300 (Acute inhalation toxicity)
according to
other: JMAFF Guidelines (2000)
GLP compliance:
Test type:
acute toxic class method

Test material

Test material form:
solid: particulate/powder

Test animals

other: Crl:WI(Han) (outbred, SPF-Quality)
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.
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.
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany) except during exposure to the test substance.
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.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
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:
Duration of exposure:
4 h
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:
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.

Results and discussion

Effect levels
Key result
Dose descriptor:
Effect level:
> 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
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.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
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.