Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
other: A four week inhalation toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Exposures were initiated on 27 October 1986 and were compl eted; on 21 November 1986.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Details on test material

Sponsor's identification: OS80020 (product as manufactured diluted to 65% in mineral oil)
Description: Brown liquid
Purity: 35% Active Ingredient (contains 65% oil)
Lot No.: Not supplied
Label: Sample 05#80020 Date 10-2-86,
Date received 03 October 1986
Storage conditions: Temperature monitored room (60 - 85°F).
Expiry date: Not supplied

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source: CD® (Sprague-Dawley) strain rats were obtained from Charles River Breeding Laboratories, Inc. Kingston, New York 12484.

- Age at study initiation: Approximately 6 (males) to 7 weeks old (females)

- Weight at study initiation: 205 to 232 g (male); 1556 to 186 g (female)

- Fasting period before study: Not applicable

- Housing: Animals were doubly-housed in suspended stainless steel wire mesh cages during the first week of the acclimation period and individually housed during the remainder of the acclimation period and all other non-exposure periods.

- Diet: was available ad libitum standard laboratory diet (Purina® Rodent Laboratory Chow #5001).

- Water: was available ad libitum via an automated watering system (Supplier: Elizabethtown Water Company).

- Acclimation period: Animals were acclimated for approximately two weeks (14 through 27 October 1986). All animals were examined by the staff veterinarian during the acclimation period.

ENVIRONMENTAL CONDITIONS

- Temperature: Monitored and recorded twice daily.
- Specified Ranges: 22 ± 3°C
- Actual range: 21-26 °C

- Humidity (%): Monitored continuously and recorded twice during exposure.
- Specified Ranges: 30 - 70%
- Actual range: 20 - 76%2

In-Life Dates: First day of treatment: 27 October 1986 - Necropsy day was 24 November 1986.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: See attached Appendix B
Details on inhalation exposure:
Test; Substance Administration and Chamber Operation (see attached Appendix B).

Route: inhalation, administered in the breathing zone of the animals as a respirable aerosol.

Justification of Route: Inhalation was chosen as the route of administration since it is a potential route of human exposure to this test substance.

Target Exposure Levels: 0 (Control), 50, 150 and 250 milligrams particulate per cubic meter of air (mg/m3).

Frequency of Exposure; Daily (6 hours per day, 5 days per week for 4 weeks).

Chamber Operation: The glass and stainless steel exposure chambers in which the animals were exposed had a total volume of 1000 liters with an effective volume of 760 liters. The airflow rate, time for air change and 99% equilibrium time are summarised as follows:

Group Airflow Air 99% Equilibrium
Rate (1pm) Change (min) Time (min)

I 213 4.7 22
II 215 4.7 21
III 211 4.7 22
IV 210 4.8 22

Exposure Procedure

An appropriate amounts of the test material was placed in an Erlenmeyer flask connected to an FMI fluid metering pump (Model #RPG-6 or RPG~20) with a 1/8" piston and an initial setting of 13, 17 and 20% for Groups II, III and. IV respectively. The test material was delivered from the flask to the inlet, of an air atomizing nozzle (Spraying Systems Co. 1/4" JSS nozzle with an #1A spray regulator (at a constant back-pressure of 20 pounds per square inch, psi} and a Dwyer flow meter via 3/8" Tygon® tubing, to the air inlet of the atomizer to generate the aerosol. The test atmosphere was directed, diluted with room air to the inlet portal of the exposure chamber which housed the animals.

The animals remained in the chamber for 30 minutes following exposure to allow the chamber air to clear using room air only.

Exposure Chamber Sampling and Monitoring:

Samples for gravimetric determinatipn of the OS80020 exposure levels were drawn from the exposure chamber using Whatman glass microfibre filter paper (Type GF/F, 3.7 cm) mounted in a Gelman filter holder. Samples were withdrawn at one and one and a half hour intervals from the normal sampling portal for 20, 20, 5 and 2 minutes at an airflow rate of 10 1pm for Groups I, II, III and IV, respectively.

Particle size Analysis

Particle size. distribution assessments were made once each week using a Delron DCI-6 cascade impactor. The mass median aerodynamic diameter (MMAD) and geometric standard deviation: were calculated based on the amount of material collected on the impactor stages (glass slides). Particle size analysis was not performed for the control group (Group I).

The nominal concentration was determined by weighing the generation apparatus containing the test substance before and after the exposure and dividing the difference in these, weights by the total volume of air delivered during exposure (volumetric flow rate times total exposure time).
An Airguide® Humidity Indicator was used to continuously monitor relative humidity and a Taylor thermometer was used to continuously monitor air temperature in the exposure chamber. Recordings of temperature and relative humidity were made four times during exposure.
























Details on analytical verification of doses or concentrations:
Chamber Monitoring Results (See attached Appendix B):

The test animals received mean gravimetric and nominal exposure concentrations of the test material as follows:

Group Test Test Mean Analytical Nominal
Substance Concentration Concentration Concentration
(mg/m3) (mg/m3) (mg/m3)

I Control 0 0.04 -
II OS80020 50 49.5 272
III OS80020 150 156 1060
IV OS80020 250 260 1360


The average concentration levels, when compared to the target levels, was considered quite acceptable. Also the day to day variability of concentrations was reasonable. The differences between nominal and analytical levels were typical for this experimental design and reflect aerosol deposition on chamber surfaces and animal fur.

Particle size analysis using the Cascade Impactor showed the average mass median diameter of the aerosol to range from 3.3 to 3.7 microns, with an average geometric standard deviation ranging from 2.0 to 2.1 . This data ,indicated that the aerosol was of a respirable size to the rat, with at least 93% of the particles less than 10 microns in diameter.

Duration of treatment / exposure:
OS80020 was administered by inhalation as an aerosol to 30 CD® (Sprague-Dawley) rats (5/sex/group) for: 6 hours per day, 5 days per week over 4 weeks, at target concentrations of 50, 150 and 250 milligrams aerosol per cubic meter of air (mg/m3). Control animals (5/sex) received air only, while in chamber. Exposures were initiated on 27 October 1986 and were completed on 21 November. 1986.
Frequency of treatment:
Once daily (5 days a week) for a six hour dosing period over a 4 week treatment period.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Control - five males and five females
Basis:
other: air only
Remarks:
Doses / Concentrations:
Group 2 - five males and five females
Basis:
other: 50 milligrams aerosol per cubic meter of air (mg/m3).
Remarks:
Doses / Concentrations:
Group 3 - five males and five females
Basis:
other: 150 milligrams aerosol per cubic meter of air (mg/m3).
Remarks:
Doses / Concentrations:
Group 4 - five males and five females
Basis:
other: 250 milligrams aerosol per cubic meter of air (mg/m3).
No. of animals per sex per dose:
five males and five females per treatment group
Control animals:
yes, sham-exposed
Details on study design:
Dose selection rationale:
- Based on previoulsy available toxicity information.

Rationale for animal assignment (if not random):
- Random

Rationale for selecting satellite groups:
- Not applicable

Post-exposure recovery period in satellite groups:
- Not applicable

Section schedule rationale (if not random):
- Random
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
- see attached Appendices A to J.

CAGE SIDE OBSERVATIONS:- Mortality, gross signs of toxicological and pharmacological effects were performed twice daily.

- All animals. were, observed for abnormalities as a group (in cage) both during the exposure, and individually following exposure.

- Detailed physical examinations were performed weekly (post-exposure on Test Days 5, 12, 19 and 26). In addition, observations were recorded once during the pre test period.

BODY WEIGHT:

- Weekly (post-exposure on Test Days 5, 12, 19 and 26). In addition, bodyweights were obtained prior to exposure on Test Day 1.

FOOD CONSUMPTION: Not applicable.

- Laboratory diet was supplied ad libitum.

FOOD EFFICIENCY: Not applicable.

WATER CONSUMPTION: Not applicable.

Water was supplied ad libitum via an automated watering system.

OPHTHALMOSCOPIC EXAMINATION: Not applicable.

HAEMATOLOGY AND CLINICAL CHEMISTRY:

Blood was obtained via veni puncture of the orbital sinus immediately prior to sacrifice.

Animals were fasted overnight prior to blood collection.

- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on all five males and five females from each test and control group prior to termination on Day 29.

URINALYSIS: Not applicable.

NEUROBEHAVIOURAL EXAMINATION:- Not applicable.

Behavioural assessment - Not applicable.

Functional Performance Tests - Not applicable.

Sensory Reactivity - Not applicable.
Sacrifice and pathology:
- see attached Appendix J.

A complete gross postmortem examinations was performed on all animals dying spontaneously as well as on those surviving to study termination. Gross postmortem examinations included examination. of the external surface, all orifices, the cranial cavity, carcass, the external and sectioned 'surfaces of the brain, and spinal cord, nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and there viscera and the cervical tissues and organs.

Selected tissues were examined from all control and high dose animals.
Other examinations:
Not applicable.
Statistics:
See atatched appendices E to I.

Bodyweights, haematology, clinical chemistry, terminal organ and body weights and organ/body weight ratios were analysed.

Mean values of all dose groups were compared to control at each time interval and statistically significant differences from control are indicated on mean table and appendices.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
RESULTS AND DISCUSSION:

Chamber Monitoring Results (see attached Appendix B).

The test animals received mean gravimetric and nominal exposure concentrations of the test material. The average concentration levels, when compared to the target levels, was considered quite acceptable. Also the day to day variability of concentrations was reasonable. The differences between nominal and analytical levels were typical for this experimental design and reflect aerosol deposition on chamber surfaces and animal fur.

Particle size analysis using the Cascade Impactor showed the average mass median aerodynamic diameter of the aerosol to range from 3.3 to 3.7 microns, with an average geometric standard deviation ranging from 2.0 to 2.1. This data indicated that the aerosol was of a respirable size to the rat, with at least 93% of the particles less than 10 microns in diameter.

Mortality (see attached Appendix C).

There was no morta1ity associated with test-materia1 exposure. A Group I male escaped from it’s caging after, the 17th exposure and was not located until the day of scheduled sacrifice when the animal was then euthanized. Also, a Group I male died during blood collection immediately prior to its scheduled sacrifice.

Clinical Observations (see attached Appendix D).

- Daily Observations during the Exposure Period:

There appeared to be test material related signs during the exposures, especially at the two higher levels. The signs were first noted after 4 to 5 exposures and consisted of red nasal discharge, matted coat and decreased activity.

- Weekly Observations during the post-exposure period:

The observations included an increased incidence of dried red nasal discharge and matted coat among the treated animals. However, the relation of these signs to exposure level was not clear. There were no significant respiratory signs seen in the test animals.

Body Weights (see attached Appendix E).

Although there were no statistically significant differences seen between control and treated groups, there was clearly a trend towards lower body weight gain in the exposed animals especially in the males at the high level.

Haematology and Clinical Chemistry (see attached Appendix F, G and H).

Several parameters in test groups were statistically different from control. These included increased hematocrit (Group II females), creatine phosphokinase (Group II and IV females) and sodium (Group IV females). However, considering that these differences were scattered throughout only one sex, and generally were small differences, the relation to treatment is questionable.

Terminal Organ and Body Weights and Organ/Body Weight Ratios (see attached Appendix I).

In both sexes, a clear dose-related trend was seen in both increased lung weights and lung ratios affecting the two higher levels. The increases were statistically significant in Group III and IV lung ratios (both sexes), Group IV lung weights (both sexes) and Group III lung weights (males).

The other organ weights and ratios were unremarkable.

Gross and Microscopic Pathology (see attached Appendix J).

A few grossly visible changes were noted. They appeared sporadic and were not considered to be related to the test article.

Microscopically, a higher incidence of accumulations of intra alveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium was seen in the lungs of the treated animals when compared to the control animals. These changes were clearly dose-related at the two higher levels, but were equivocal at the lower level. Other microscopic findings in the lungs and other tissues did not appear to be related to the test article.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The repeated inhalation exposure of OS80020 (product as manufactured diluted to 65% in mineral oil) by aerosol atmospheres for a period of twenty-eight consecutive days at target concentrations of 50, 150 and 250 mg/m3 produced treatment-related changes at all dose levels and on this basis a ‘No Observed Effect Level’ (NOEL) could not be established.

The change observed at a target dose concentration of 50 mg/m3 was considered not to be indicative of a serious adverse effect of treatment and on this basis may be regarded as a “No Observed Adverse Effect Level” (NOAEL).

Executive summary:

In this study, OS80020 (product as manufactured diluted to 65% in mineral oil) was administered to assess the toxic effects by inhalation of the test item as an aerosol to 30 rats (5/sex/group) for 6 hours per day, 5 days per week for 4 weeks at target concentrations of 50, 150 and 250 milligrams aerosol per cubic meter of air (mg/m3). Control animals (5/sex) received; air only, while in chamber.

 

Exposures were initiated on 27 October 1986 and were completed on 21 November 1986. Exposure levels were monitored gravimetrically four, times per chamber per day. Particle size distribution measurements were made once each week using a Delron DCI-6 Cascade Impactor. 

 

Physical observations for abnormal signs were made during exposure for all animals. Detailed physical examinations were conducted weekly on all animals. Body weight measurements were recorded weekly and once during the pre test period, on Test Day 1; body weight measurements were also obtained just prior to sacrifice on Test Day 29. Just prior to sacrifice, blood samples were obtained for haematology and clinical chemistry then all animals were sacrificed, selected organs were weighed and organ/body weight ratios were calculated. A complete gross post mortem examination was conducted on all animals followed by microscopic examination of selected tissues.

 

The cumulative mean analytical exposure concentrations as determined gravimetrically were 49.5, 156 and 260 mg/m3 OS80020, with an average nominal concentration of 272, 1060 and 1360 mg/m3OS80020 for the low, mid and high dose groups, respectively. Particle size distribution determinations indicated the test aerosol atmosphere was respirable to the rat.

 

All animals survived the duration of the study.

 

Physical observations during the exposures included red nasal discharge, matted coat and decreased activity at the two higher levels.

 

No significant respiratory signs were noted during the weekly observations; however increased incidence of dried red nasal discharge and matted coat was seen among the treated animals.

 

Body weight measurements indicated a trend towards lower weight gain, especially in the high level males. However, this difference was not statistically significant.

 

Haematology and clinical chemistry parameters were generally not indicative of any test-material effect.

 

Terminal organ weight and organ body weight ratios were clearly indicative of a respiratory effect with significant dose related increases in lung weights and ratios at the mid and high levels. Other organ weights and ratios were unremarkable.

 

Gross post mortem evaluations were unremarkable. However, a higher incidence was seen microscopically in, the mid and high level lungs of accumulation of intra alveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium. Other microscopic findings were not considered test-article related.