Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 - 27 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.436, "Acute Inhalation Toxicity - Acute Toxic Class Method", September 2009.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
off-white powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han) - (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
Animal source: Charles River Deutschland, Sulzfeld, Germany.
Young adult animals were selected (approximately 10 weeks old). Animals used within the study were of approximately the same age and body weight variation did not exceed +/- 20% of the sex mean. At least prior to exposure animal health was inspected. It was ensured that the animals were hwalthy and without any abnormality that might affect the study integrity.

The temperature and relative humidity were measured with a humidity and temperature indicator (E+E Elektronik, Engerwitzdorf, Austria) and were recorded after the animals were placed in the experimental set-up and at 30 minute intervals after initiation of the exposure. The probe was inserted in a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The temperature of the atmosphere was between 21.2 and 22.1oC and relative humidity was between 12 and 18%. These conditions were considered appropriate for this relatively short 4 hours exposure duration.

Animal Husbandry
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
Group housing of three 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. During this period, animals were housed at maximally five animals per cage per sex as described above.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaeten 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.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.3 - <= 4.9 µm
Geometric standard deviation (GSD):
>= 1.8 - <= 2
Remark on MMAD/GSD:
At 1 mg/l concentration: 4.9 μm (gsd 1.8) at both occasions
At 5 mg/l concentration: 4.9 μm (gsd 2.0) and 3.3 μm (gsd 1.8)
Details on inhalation exposure:
The design of the exposure chamber is based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber (volume approximately 150 mL) consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet. The animals were placed in restraining tubes and connected to the animal ports. The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal. All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.

The test atmosphere generation was based on the method developed during extensive trial generations. Trial generation results showed that the test substance was not volatile and the test atmosphere consisted mainly of aerosol with a negligibly small (if any) vapor part. The generation was performed at the technically maximum attainable concentration.

The test substance formulation was placed on a magnetic stirrer and was transferred to a stainless steel nebulizer (type 156.000.16.16, Lechler, Metzinger, Germany) by means of a rotating pump (type VL500 digit, VERDER Lab Tec GmbH & Co. KG, Haan, Germany). The nebulizer was mounted under a vertical elutriator in which large droplets were allowed to settle. The formulation was nebulized with the use of pressurized air with a mean total airflow of 17.3 L/min. From the elutriator the test atmosphere was passed through the exposure chamber.

For the exposure to 1 mg/L, the primary aerosol was diluted with pressurized air before it entered the exposure chamber and the resulting mean total airflow was 40 L/min.
For the exposure to 5 mg/L, the mean total airflow was 15 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.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric determination
Duration of exposure:
ca. 4 h
Concentrations:
Target concentrations were based on the cut off concentration values specified in the UN and EC classification guidelines. The study started with the exposure of three animals of each sex for 4 hours to a target concentration of the test substance of 1 mg/L. Based on the results, three animals of each sex were exposed to the highest target concentration of 5 mg/L.

For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.2 ± 0.04mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 20 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 6%. The concentration measurements equally distributed over time showed that the substance was sufficiently stable. The variations in concentration were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. By calculating the time-weighted mean concentration, the effects of these variations were taken into account resulting in an actual reflection of the mean exposure concentration over time.

For the 5 mg/L exposure group, the time-weighted mean actual concentration was 5.6 ± 0.15mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 158 mg/L. The generation efficiency (ratio of actual and nominalconcentration) was 0.6%. The concentration measurements equally distributed over time showed that the substance was sufficiently stable. The variations in concentration were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. The generation was interrupted at three occasions in order to remove test substance deposits from the system or to refill the dust feeder with test substance. The generation time was elongated with 13 minutes in order to achieve an actual exposure time of 240 minutes.
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
Observations
Mortality / Viability: Twice daily
Clinical signs during exposure: Three times during exposure for mortality, behavioural signs of distress and effects on respiration
Clinical signs after exposure: On Day 1, one and three hours after exposure and once daily thereafter until Day 15. The clinical signs were graded according to fixed scales and the time of onset, degree and duration were recorded.
Body weights: Days 1 (pre-administration), 2, 4, 8 and 15
Necropsy: All animals were euthanatized at the end of the observation period by an intraperitoneal injection with Euthasol ® (AST Farma BV, Oudewater, The Netherlands). All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract.

Results and discussion

Preliminary study:
No
Effect levelsopen allclose all
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air (nominal)
Based on:
test mat. (total fraction)
Exp. duration:
4 h
Key result
Sex:
male/female
Dose descriptor:
LC50 cut-off
Effect level:
> 12.5 mg/L air (nominal)
Based on:
test mat. (total fraction)
Exp. duration:
4 h
Mortality:
No mortality occurred at 1 and 5 mg/l.
Clinical signs:
other: At 1 mg/L, no clinical signs of systemic toxicity were noted. At 5 mg/L, no clinical signs were seen during exposure (not presented in the table). After exposure, lethargy, hunched posture, and piloerection were observed in the animals. Laboured respirati
Body weight:
Overall mean body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.
Gross pathology:
No test substance related abnormalities were found at macroscopic post mortem examination of the animals.

Pelvic dilation of the kidney, as found in one male exposed to 5 mg/L, is occasionally seen among rats of this age and strain and was therefore considered not related to treatment.

Any other information on results incl. tables

See attached pdf document.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The inhalatory LC50, 4h value of BMS-214702-01 in Wistar rats was established to exceed 5mg/L. According to the OECD 436 test guideline, the LC50, 4h cut-off value was considered to exceed 12.5 mg/L.

Based on these results BMS-214702-01 does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).
Executive summary:

Assessment of acute inhalation toxicity with BMS-214702-01 in the rat (acute toxic class method) (nose-only).

The study was carried out based on the guideline described in Organisation for Economic Cooperation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.436, "Acute Inhalation Toxicity - Acute Toxic Class Method", September 2009.

BMS-214702-01 was administered as a dust by nose-only inhalation for 4 hours to two groups of three males and three females. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15. Macroscopic

examination was performed after terminal (Day 15).

For the 1 mg/L exposure group, the time-weighted mean actual concentration was 1.2 ± 0.04 mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 20 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 6%. The concentration measurements equally distributed over time showed that the substance was sufficiently stable.

For the 5 mg/L exposure group, the time-weighted mean actual concentration was 5.6 ± 0.15 mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 158 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 0.6%. The concentration measurements equally distributed over time showed that the substance was sufficiently stable.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during the each exposure period. At 1 mg/L, the MMAD was 4.9 µm (gsd 1.8) at both occasions. At 5 mg/L, the MMAD was 4.9 µm (gsd 2.0) and 3.3 µm (gsd 1.8).

The MMAD just exceeded the recommended range of 1 - 4 µm at three occasions. There was no evidence for substance deposition in the upper airways due to the larger size. Since it is generally known that good distribution throughout the lung requires particles with an aerodynamic diameter between 1 and 5 μm, it can be assumed that sufficient deposition in the lower respiratory tract occurred during the exposure. At 5 mg/L, the MMAD became smaller during the exposure since the test material had to be retrieved from the cyclone, grinded and used again due to the limited availability of fresh test substance. It was considered that the study outcome was not affected.

No mortality occurred at 1 and 5 mg/L. At 1 mg/L, no clinical signs of systemic toxicity were noted. At 5 mg/L, no clinical signs were seen during exposure (not presented in the table). After exposure, lethargy, hunched posture, and piloerection were observed in the animals. Laboured respiration and ptosis were seen for the males and watery discharge and chromodacryorrhoea was seen for the females. The animals had recovered from the signs by Day 6.

Overall mean body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.

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

The inhalatory LC50, 4h value of BMS-214702 -01 in Wistar rats was established to exceed 5 mg/L According to the OECD 436 test guideline, the LC50,4h cut-off value was considered to exceed 12.5 mg/L.

Based on these results BMS-214702-01 does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all

amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).