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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to OECD 436, in full comformance with GLP. Gravimetric and analytical verification of test article concentrations in nose-only aerosol exposures for 4-hours. analytical verification of MMAD (2-3 u).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: Tert-butyl Peroxybenzoate
Description: Colorless liquid
CAS Number: 614-45-9
Batch Number: 0903225181
Purity: 99.2%
Expiry Date (Retest Date): 22-Mar-2010
Storage Conditions: In the refrigerator (5 ± 3°C), protected from direct
sunlight.

Test animals

Species:
rat
Strain:
other: HanRcc:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Husbandry
Room Numbers: Animal room number 306 and 307 (Group 1) and
307 and 315 (Group 2), Harlan Laboratories Ltd,
Füllinsdorf.
Conditions: Optimal laboratory conditions behind a barrier
system. Air-conditioned with 10 - 15 air changes per
hour, continuously monitored environment with
temperature range of 22 ± 3 °C, a relative humidity
range of 30 - 70% and a 12 hour fluorescent light /
12 hour dark cycle. A radio program was played
during most of the light period.
Accommodation: Animals were housed in groups of 3 of the same sex
in Makrolon® type-IV cages with wire mesh tops
and standard softwood bedding ("Lignocel" J.
Rettenmaier & Söhne GmbH & Co KG, 73494
Rosenberg / Germany, imported by Provimi Kliba
AG, 4303 Kaiseraugst / Switzerland).
Diet: Animals had ad libitum access to a pelleted standard
Teklad rat maintenance diet (Provimi Kliba AG,
4303 Kaiseraugst, Switzerland) batch no. 82/09
except during the period when the animals were
restrained in exposure tubes. Results of the analyses
for contaminants and their limits of acceptability are
archived at Harlan Laboratories Ltd.
Water: Community tap water from Füllinsdorf ad libitum in
water bottles, except during the period when they
were restrained in exposure tubes. Results of
representative analyses for contaminants are
archived at Harlan Laboratories Ltd.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
Treatment
Room: Inhalation laboratory no. 310, Harlan Laboratories
Ltd., Füllinsdorf
Method: Inhalation by nose-only, flow-past exposure.
Rationale for Method: Inhalation is a possible route of human exposure.
Frequency of Administration: Single, 4-hour exposure period.
Allocation and Target AerosolConcentration:
Males Females Target AerosolConcentration(mg/L air)
Group 1 1-3 4-6 slightly above 5
Group 2 7-9 10-12 slightly above 1

Rationale for Aerosol Concentration:
Group 1:
The target concentration of slightly above 5 mg/L air
for 4 hours is the recommended concentration for a
limit test (OECD 436, “Acute Inhalation Toxicity”).
Group 2:
The target concentration of 1 mg/L air for 4 hours is
the recommended concentration according to annex
3 d (OECD 436, “Acute Inhalation Toxicity”).
Duration of Observation Period: 14 days

Inhalation Exposure System
Inhalation exposure was performed using a system similar to that originally described by Sachsse
et al. (see References (1) and (2)). The animals were confined separately in restraint tubes which
were positioned radially around the flow-past, nose-only exposure chamber as described by
Cannon et al. (see References (3)). The design of this chamber is based upon the fluid dynamic
modeling of the test aerosol flow.
The exposure system ensured a uniform distribution and provided a constant flow of test material
to each exposure tube. The flow of air at each tube was 1.0 L/min, which is sufficient to
minimize re-breathing of the test atmosphere as it is more than twice the respiratory minute
volume of a rat.
Before commencement of the exposure of the groups, technical trials were conducted (without
animals) using the inhalation system foreseen for the study. The technical trials were conducted
using established procedures based on GLP, but were not inspected by the Harlan Laboratories
Ltd. Quality Assurance. Technical trial data are retained in the raw data.

Test Aerosol Generation
The test atmosphere was generated using a Hudson nebulizer connected to a syringe pump. The
polyethylene injector inside the nebulizer was replaced by a stainless steel injector.

Exposure System Monitoring
The aerosol concentration, the particle size distribution, relative humidity, temperature and
oxygen concentration were measured on test aerosol samples taken at a representative exposure
port.

Nominal Determination of Aerosol Concentration
The test item usage was measured by weighing the syringe and nebulizer reservoir containing the
test item before and after exposure to determine the quantity of test item used. The weight used
was then divided by the total air-flow volume to give the nominal concentration.

Gravimetric Determination of Aerosol Concentrations
Gravimetric determinations of aerosol concentration were performed four times during each
exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47
mm in-line stainless steel filter sampling device. The filters were weighed before and
immediately after sampling using a calibrated balance. The test aerosol concentration was
calculated from the amount of test item present on the filter and the sample volume.

Chemical Determination of Aerosol Concentrations
Chemical determinations of aerosol concentration were performed four times during each
exposure.The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm inline
stainless steel filter sampling device. The filters were transferred into appropriate labeled
vials, forwarded in a cool box to the responsible scientist for analytical chemistry and stored at 2-
8 °C until analysis. The samples were analyzed using a HPLC method supplied by the Sponsor.

Particle Size Distribution and Mass Median Aerodynamic Diameter
The particle size distribution was measured gravimetrically three times during each exposure
using a 7 stage cascade Mercer Impactor (Model 02-130, In-Tox. Products Inc., Albuquerque,
New Mexico, U.S.A.). Mass Median Aerodynamic Diameters and Geometric Standard
Deviations were calculated on the basis of the results from the impactor, using Microsoft Excel
Software. The target range for the Mass Median Aerodynamic Diameter was 1 to 4 μm.

Oxygen Concentration
The oxygen concentration of the test atmosphere was measured continuously during each
exposure using a calibrated device. The results were recorded manually and are reported at 30
minute intervals from the start of exposure. The oxygen concentration was maintained above
19% during each exposure period.

Relative Humidity / Temperature
The temperature and relative humidity of the test atmosphere was measured continuously during
each exposure using a calibrated device. The results were recorded manually and are reported at
30 minute intervals from the start of exposure.

Airflow Rate
The actual airflow rate through the exposure chamber was recorded at approximately 30 minute
intervals from the start of the inhalation exposure.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
1.01 mg/L and 4.9 mg/L
No. of animals per sex per dose:
3M & 3F
Control animals:
no
Details on study design:
Treatment
Room: Inhalation laboratory no. 310, Harlan Laboratories
Ltd., Füllinsdorf
Method: Inhalation by nose-only, flow-past exposure.
Rationale for Method: Inhalation is a possible route of human exposure.
Frequency of Administration: Single, 4-hour exposure period.
Allocation and Target AerosolConcentration:
Males Females Target AerosolConcentration(mg/L air)
Group 1 1-3 4-6 slightly above 5
Group 2 7-9 10-12 slightly above 1

Rationale for Aerosol Concentration:
Group 1:
The target concentration of slightly above 5 mg/L air
for 4 hours is the recommended concentration for a
limit test (OECD 436, “Acute Inhalation Toxicity”).
Group 2:
The target concentration of 1 mg/L air for 4 hours is
the recommended concentration according to annex
3 d (OECD 436, “Acute Inhalation Toxicity”).
Duration of Observation Period: 14 days

Inhalation Exposure System
Inhalation exposure was performed using a system similar to that originally described by Sachsse
et al. (see References (1) and (2)). The animals were confined separately in restraint tubes which
were positioned radially around the flow-past, nose-only exposure chamber as described by
Cannon et al. (see References (3)). The design of this chamber is based upon the fluid dynamic
modeling of the test aerosol flow.
The exposure system ensured a uniform distribution and provided a constant flow of test material
to each exposure tube. The flow of air at each tube was 1.0 L/min, which is sufficient to
minimize re-breathing of the test atmosphere as it is more than twice the respiratory minute
volume of a rat.
Before commencement of the exposure of the groups, technical trials were conducted (without
animals) using the inhalation system foreseen for the study. The technical trials were conducted
using established procedures based on GLP, but were not inspected by the Harlan Laboratories
Ltd. Quality Assurance. Technical trial data are retained in the raw data.

Test Aerosol Generation
The test atmosphere was generated using a Hudson nebulizer connected to a syringe pump. The
polyethylene injector inside the nebulizer was replaced by a stainless steel injector.

Exposure System Monitoring
The aerosol concentration, the particle size distribution, relative humidity, temperature and
oxygen concentration were measured on test aerosol samples taken at a representative exposure
port.

Nominal Determination of Aerosol Concentration
The test item usage was measured by weighing the syringe and nebulizer reservoir containing the
test item before and after exposure to determine the quantity of test item used. The weight used
was then divided by the total air-flow volume to give the nominal concentration.

Gravimetric Determination of Aerosol Concentrations
Gravimetric determinations of aerosol concentration were performed four times during each
exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47
mm in-line stainless steel filter sampling device. The filters were weighed before and
immediately after sampling using a calibrated balance. The test aerosol concentration was
calculated from the amount of test item present on the filter and the sample volume.

Chemical Determination of Aerosol Concentrations
Chemical determinations of aerosol concentration were performed four times during each
exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm inline
stainless steel filter sampling device. The filters were transferred into appropriate labeled
vials, forwarded in a cool box to the responsible scientist for analytical chemistry and stored at 2-
8 °C until analysis. The samples were analyzed using a HPLC method supplied by the Sponsor.

Particle Size Distribution and Mass Median Aerodynamic Diameter
The particle size distribution was measured gravimetrically three times during each exposure
using a 7 stage cascade Mercer Impactor (Model 02-130, In-Tox. Products Inc., Albuquerque,
New Mexico, U.S.A.). Mass Median Aerodynamic Diameters and Geometric Standard
Deviations were calculated on the basis of the results from the impactor, using Microsoft Excel
Software. The target range for the Mass Median Aerodynamic Diameter was 1 to 4 μm.

Oxygen Concentration
The oxygen concentration of the test atmosphere was measured continuously during each
exposure using a calibrated device. The results were recorded manually and are reported at 30
minute intervals from the start of exposure. The oxygen concentration was maintained above
19% during each exposure period.

Relative Humidity / Temperature
The temperature and relative humidity of the test atmosphere was measured continuously during
each exposure using a calibrated device. The results were recorded manually and are reported at
30 minute intervals from the start of exposure.

Airflow Rate
The actual airflow rate through the exposure chamber was recorded at approximately 30 minute
intervals from the start of the inhalation exposure.
Statistics:
none

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
male/female
Dose descriptor:
LC100
Effect level:
4.9 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Key result
Sex:
male/female
Dose descriptor:
LC0
Effect level:
1.01 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
6/6 at 4.9 mg/L
Clinical signs:
Mortality and Clinical Signs

Group 1:
One male and one female were found dead on day 2 of the observation period. The remaining
two males were found dead on day 3. One female was found dead on day 11. The remaining
female was killed in extremis on day 15.
Salivation was recorded during and up to immediately after exposure in all animals. Decreased
activity, ruffled fur and labored breathing were observed in all animals after exposure on day 1
and day 2. Ruffled fur persisted up to day 5 in one female. Decreased activity and ruffled fur
were recorded up to day 6 in the other surviving female. Tachypnea was recorded on day 5 and 6
and decreased activity, hunched posture, ruffled fur and tachypnea were seen from day 13 until
necropsy in this female.

Group 2:
All animals survived the scheduled observation period.
Salivation was recorded during and/or immediately after exposure in all animals. Ruffled fur was
observed in all animals on day 1 and 2 of the observation period. Effects on breathing, like
labored breathing or tachypnea, were seen in all animals on the day of exposure up to day 2.
Decreased activity was recorded in all males 1 hour after exposure end. There were no clinical
signs from day 3 onwards.


Body weight:
Group 1:
From test day 1 to test day 2, marked body weight loss was noted in all surviving animals.
Further body weight loss was recorded in the surviving females until day 4. Thereafter body
weight gain was recorded in these animals although one of these females was found dead on day
9. The other female showed a further body weight loss from day 8 to day 15.
Group 2:
From test day 1 to test day 2, slight body weight loss was noted in all males. Stagnation of body
weight was recorded in two females. Thereafter normal body weight development was recorded
in all males and two females. Stagnation of body weight was observed in one female up to day 8.
Gross pathology:
Group 1:
Incompletely collapsed lung, dark red or reddish discoloration of the lung or the thymus, were
seen in several animals during necropsy.
Group 2:
There were no macroscopic findings.

Applicant's summary and conclusion

Interpretation of results:
Toxicity Category IV
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Treatment of HanRcc:WIST(SPF) rats with Tert-butyl Peroxybenzoate at a concentration of 4.9
and 1.01 mg/L for 4 hours resulted in effects on body weight and clinical signs such as ruffled
fur, decreased activity and breathing problems.
Incompletely collapsed lung and dark red discoloration of the lung, seen in the female that had to
be killed in extremis after exposure at 4.9 mg/L air, may be related to treatment with the test item.
All other macroscopic findings were considered to be due to the premature death of the animals.
Exposure at 4.9 mg/L air caused the premature death of 6 animals. The remaining female was
killed on day 15 due to marked body weight loss during the observation period.
In conclusion, the LC50 of Tert-butyl Peroxybenzoate obtained in this study was estimated to be
between 4.9 and 1.01 mg/L air (chemically determined mean aerosol concentration). Due to this
LC50 range the test item was classified in category 4 (GHS). There was no indication of relevant
sex-related differences in toxicity of the test item.
Executive summary:

Two groups of three male and three female albino rats [HanRcc:WIST(SPF)] were exposed by nose-only, flow-past inhalation for four hours to the test item at a chemically determined mean concentration of 4.9 and 1.01 mg/L air, respectively. All surviving animals were observed for clinical signs and mortality during the inhalation exposure and the observation period of 14 days. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 2, 4, 8 and 15 before necropsy. On day 15 all surviving animals were sacrificed and necropsied. The ranges of aerosol concentration, temperature, relative humidity, oxygen content and airflow rate measured during the exposure were considered to be satisfactory for a study of this type. In addition, the test item was considered to be respirable to rats. Group 1: One male and one female were found dead on day 2 of the observation period after exposure at 4.9 mg/L air. The remaining two males were found dead on day 3. One female was found dead on day 11. The remaining female was killed for humane reasons on day 15. At 4.9 mg/L air salivation was recorded during up to immediately after exposure in all animals. Decreased activity, ruffled fur and labored breathing were observed in all animals after exposure on day 1 and day 2. These signs persisted partly up to day 6. Decreased activity, hunched posture, ruffled fur and tachypnea were seen from day 13 until necropsy in this female. Marked effects on body weights were recorded in all animals. Group 2: All animals survived the scheduled observation period at 1.01 mg/L air. Salivation was recorded during and/or immediately after exposure in all animals. Decreased activity was recorded in all males 1 hour after exposure end. Ruffled fur and effects on breathing were recorded in most of the animals on days 1 and 2. There were no clinical signs from day 3 onwards. Effects on body weights were recorded in all animals. There were no macroscopic findings in groups 1 and 2 that were considered to be related to treatment with the test item. In conclusion, the LC50 of tert-butyl perbenzoate (CAS# 614-45-9) obtained in this study was estimated to be between 1.01 and 4.9 mg/L air (chemically determined mean aerosol concentration). Due to this LC50 range the test item was classified in category 4 (GHS). There was no indication of relevant sex-related differences in toxicity of the test item.