Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data to permit a meaningful evaluation of study results
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Principles of method if other than guideline:
A series of single 4-hour inhalation whole-body exposures was performed in which Zoldine ZE-P-1601 was administered as an aerosol in the breathing zone of Sprague-Dawley rats in order to determine the median lethal concentration (LD50)
GLP compliance:
no
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zoldine ZE (BIOBANTMCS-1246 Antimicrobial)
- Physical state: Dark yellow clear liquid
- Analytical purity: 96.84% determined by gas chromatography
- Lot/batch No.: # 1
- Stability under test conditions: Stable at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MASS
- Age at study initiation: Not specified
- Weight at study initiation: Males 201-299 grams, Females 203-249 grams
- Fasting period before study: Not specified
- Housing: Individually housed in elevated, stainless steel and wire mesh cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
A 250 ml reservoir containing the test material was connected to an FMI fluid metering pump (Model #RPG-20). The test material was fed directly into an air atomizing nozzle (spraying systems company; ¼” J nozzle) and dry air at a flow rate of 19 liters per minute was passed through the atomizer to generate the aerosol. The test atmosphere was directed, undiluted, into a 100-liter Plexiglas exposure chamber, which housed the animals. The chamber concentration for each exposure group was achieved by changing the FMI pump setting.
- Exposure chamber volume:
100-liter
- Method of holding animals in test chamber:
The animals were individually housed in elevated; stainless steel and wire mesh cages.
- System of generating particulates/aerosols:
The test material was fed directly into an air atomizing nozzle (spraying systems company; ¼” J nozzle) and dry air at a flow rate of 19 liters per minute was passed through the atomizer to generate the aerosol.
- Method of particle size determination:
Particle size was measured for each exposure using a cascade impactor, and the mass median aerodynamic diameter (MMAD) was calculated.


TEST ATMOSPHERE
- Brief description of analytical method used:
During each exposure chamber air samples were collected using a glass fiber filter at approximately hourly intervals at a rate of 4.9 liters per minute for 30 seconds. The amount of material collected as an aerosol was determined gravimetrically by weighing the filter holder and filter paper before and after sampling and dividing the difference in weights by the sample volume.
- Samples taken from breathing zone: yes

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
0, 1.6, 2.4, 2.6, 3.3, 4.3 mg/L
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
Single, four-hour whole-body aerosol exposures were conducted with 5 male and 5 female Sprague-Dawley rats in order to determine the median lethal dose. Rats were obtained from a commercial supplier. They were group housed during a 14-day acclimation period to the laboratory. They were housed individually and identified by metal ear tag after assignment to study. They were given food and water ad libitum during their stay in the testing facility.

Chamber monitoring was accomplished by gravimetric analysis (glass fiber filter) periodically throughout the exposure. Particle size was measured for each exposure using a cascade impactor, and the mass median aerodynamic diameter was calculated. Chamber temperature and relative humidity were maintained within reasonable ranges for a rat and recorded hourly, and animal observations were recorded during the exposure. They were observed daily until necropsy for signs of toxicity.

The aerosol was generated using an air atomizing nozzle fed with test material using an FMI pump. The test atmosphere was directed undiluted into the exposure chamber (100 liters) housing the animals. The variations in chamber concentration was achieved by changing the pump settings. A nominal concentration was calculated based on average air flow, length of exposure, chamber volume, and the amount of test material consumed by the system.

Animals were weighed on days 0, 1, 2, 4, 7, and 14. They were sacrificed on day 14 and subjected to gross pathological examination. Surviving animals were also subjected to histopathological examination on kidneys, liver, lungs, and gross lesions.

Particle size exceeded the guideline-recommended 1-4 micron MMAD in 3 of 5 exposures. The exposure was conducted whole-body, which may have introduced an oral or dermal exposure route. Animals were noted with discolored fur.
Statistics:
The calculation of median lethal concentration and 95% confidence intervals was performed by the method of Litchfield and Wilcoxon.

Results and discussion

Preliminary study:
Two of the Zoldine-ZE exposures (1.6 mg/l and 2.6 mg/l) had MMAD values of 4 microns or less. Both exposures were below the calculated LC50 (1.6 mg/l - 0/5 female, 0/5 male; 2.6 mg/l - 2/5 female, 1/5 male). Acute toxicity of the test material was concentration dependent, and female rats were more sensitive than males.
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
3.1 mg/L air
Remarks on result:
other: 95% CI, 2.7-3.5 mg/L
Mortality:
All test material-dependent deaths occurred on the day of or the day after exposure. In the absence indications of systemic injury, the rapid death suggests severe irritation/injury to the respiratory tract. Histopathological examination of the lungs of the lungs of all rats failed to show treatment-related pulmonary injury. This suggests irritation/injury to the upper respiratory tract, however, no examination of the nasal airways was conducted.
Clinical signs:
At exposures of 2.6 mg/L and higher, the animals were noted huddled with eyes closed. The fur was matted and soiled. Rats were noted with increased lacrimation and perinasal soiling, salivation, and periocular soiling. Hunched posture, loss of or reduced righting reflex, ataxia, decreased muscle tone, shallow and labored breathing, and gasping. Ocular abnormalities (corneal opacities or irregularities, dull eyes, dilated pupils, and swollen eyelids) were also noted. These symptoms resolved slightly during the observation period. Many animals exposed to 2.4 mg/L and higher were additionally observed with red extremities or paws, which resolved during the post-exposure period.
Body weight:
Body weights for surviving animals recovered to pre-exposure weights by day 4 (males) or day 7 (females). Body weights during the second week were unremarkable for all test animals, and were comparable to the control animals at termination.
Gross pathology:
Lung discoloration was the only macroscopic finding unique to pathology. Microscopic lung findings were similar in treated and control animals. The main changes were a thickening of the alveolar septa by lymphocytes and macrophages, accompanied by cellular exudation of macrophages and neutrophils in the aveolar lumen. The alterations were orientated around the respiratory bronchioles and surrounding alveoli. Alveoli hemorrhage was focal and mild in a few animals, and was considered to be typical of a low degree of interstitial pneumonia, a common incidental finding in the laboratory rat. Hepatic and renal findings were infrequent and spontaneous, and not considered related to treatment.
Other findings:
Particle size ranged from a low of 3.9 to a high of 4.7 microns (MMAD). Particle size was minimally affected by chamber aerosol concentration which suggests it reflects the inherent particle size of the test material.

Any other information on results incl. tables

Acceptable temperatures and relative humidities were recorded during the exposures (68-78F and 62-90%, respectively).  Particle size distributions ranged from 3.9-4.7 microns MMAD.

Exposure
       Particle             Mortality
Level
            Size
(mg/L)
        (microns)             
0
                n.a.                 0/10
1.6
              3.9                  0/10
2.4
              4.4                  1/10
2.6
              4.0                  3/10
3.3
              4.2                  7/10
4.3
              4.7                  8/10

Based on mortality data, the LC50 of the test material was reported to be 3.1 mg/L (95% CI, 2.7-3.5 mg/L).

At exposures of 2.6 mg/L and higher, the animals were noted huddled with eyes closed.
  The fur was matted and soiled.  Rats were noted with increased lacrimation and perinasal soiling, salivation, and periocular soiling.  Hunched posture, loss of or reduced righting reflex, ataxia, decreased muscle tone, shallow and labored breathing, and gasping.  Ocular abnormalities (corneal opacities or irregularities, dull eyes, dilated pupils, and swollen eyelids) were also noted.  These symptoms resolved slightly during the observation period.  Many animals exposed to 2.4 mg/L and higher were additionally observed with red extremities or paws, which resolved during the post-exposure period.

Body weights for surviving animals recovered to pre-exposure weights by day 4 (males) or day 7 (females).
  Body weights during the second week were unremarkable for all test animals, and were comparable to the control animals at termination.

Lung discoloration was the only macroscopic finding unique to pathology.
  Microscopic lung findings were similar in treated and control animals.  The main changes were a thickening of the alveolar septa by lymphocytes and macrophages, accompanied by cellular exudation of macrophages and neutrophils in the aveolar lumen.  The alterations were orientated around the respiratory bronchioles and surrounding alveoli.  Alveoli hemorrhage was focal and mild in a few animals, and was considered to be typical of a low degree of interstitial pneumonia, a common incidental finding in the laboratory rat.  Hepatic and renal findings were infrequent and spontaneous, and not considered related to treatment.

Applicant's summary and conclusion

Interpretation of results:
sligthly toxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In summary, in light of the lack of test material induced pulmonary injury, the effect of the particle size being slightly greater than 4 microns (MMAD) on the observed toxicity and estimation of the LC50 is believed to be minimal. LC50 was found to be 3.1 mg/L (95% CI, 2.7-3.5 mg/L).
Executive summary:

Single, four-hour whole-body aerosol exposures were conducted with 5 male and 5 female Sprague-Dawley rats in order to determine the median lethal dose. Rats were obtained from a commercial supplier. They were group housed during a 14-day acclimation period to the laboratory. They were housed individually and identified by metal ear tag after assignment to study. They were given food and waterad libitumduring their stay in the testing facility.

Chamber monitoring was accomplished by gravimetric analysis (glass fiber filter) periodically throughout the exposure. Particle size was measured for each exposure using a cascade impactor, and the mass median aerodynamic diameter was calculated. Chamber temperature and relative humidity were maintained within reasonable ranges for a rat and recorded hourly, and animal observations were recorded during the exposure. They were observed daily until necropsy for signs of toxicity.

The aerosol was generated using an air atomizing nozzle fed with test material using an FMI pump. The test atmosphere was directed undiluted into the exposure chamber (100 liters) housing the animals.  The variations in chamber concentration were achieved by changing the pump settings. A nominal concentration was calculated based on average air flow, length of exposure, chamber volume, and the amount of test material consumed by the system.

Animals were weighed on days 0, 1, 2, 4, 7, and 14. They were sacrificed on day 14 and subjected to gross pathological examination. Surviving animals were also subjected to histopathological examination on kidneys, liver, lungs, and gross lesions.

The calculation of median lethal concentration and 95% confidence intervals was performed by the method of Litchfield and Wilcoxon. Two of the Zoldine-ZE exposures (1.6 mg/l and 2.6 mg/l) had MMAD values of 4 microns or less. Both exposures were below the calculated LC50(1.6 mg/l - 0/5 female, 0/5 male; 2.6 mg/l - 2/5 female, 1/5 male). Acute toxicity of the test material was concentration dependent, and female rats were more sensitive than males.

 All test material-dependent deaths occurred on the day of or the day after exposure. In the absence indications of systemic injury, the rapid death suggests severe irritation/injury to the respiratory tract. Histopathological examination of the lungs of the lungs of all rats failed to show treatment-related pulmonary injury. This suggests irritation/injury to the upper respiratory tract, however, no examination of the nasal airways was conducted.

 

In summary, in light of the lack of test material induced pulmonary injury, the effect of the particle size being slightly greater than 4 microns (MMAD) on the observed toxicity and estimation of the LC50is believed to be minimal. LC50 was found to be 3.1 mg/L (95% CI, 2.7-3.5 mg/L).