Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994
Reference Type:
other company data
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, Stone Ridge, New York, USA
- Age at study initiation: 21-23 days, at receipet
- Weight at study initiation: 50-75 g, at receipt
- Fasting period before study: not reported
- Housing: suspended wire mesh cage
- Diet (e.g. ad libitum): Purina certified rodent chow 5002; withheld during exposure
- Water (e.g. ad libitum): ad libitum; withheld during exposure
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.1 - 23.4 degrees C
- Humidity (%): 67.8 - 74.6%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark


IN-LIFE DATES: From: May 25, 1993 To: June 22, 1993

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable- vapor study
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1250 L stainless steel, glass and Plexiglass chambers
- Method of holding animals in test chamber: PVC nose-only restraining tubes
- Source and rate of air: Compressed air at 0.25 to 32 L/min
- Method of conditioning air: no data
- System of generating particulates/aerosols: study conducted with vapor, not aerosol
- Temperature, humidity, pressure in air chamber: 23.1-23.4 degrees C; 67.8 - 74.6%; pressure not reported
- Air flow rate: air flow rate adjutsted to 99% of the maximum vapor concentration
- Air change rate: no reported
- Method of particle size determination: study conducted with vapor, not aerosol
- Treatment of exhaust air: not reported


TEST ATMOSPHERE
- Brief description of analytical method used: an impinger located outside each chamber containing 8-12 mL of propylene glycol placed in an ice bath
- Samples taken from breathing zone: yes


VEHICLE (if applicable) no vehicle
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to determine the analytical concentration of the tetst material in the chambers, one impinger located outside wach chamber was used for sampling. The impingers contained 8-12 mL of propylene glycol and were placed in an ice bath. The amount of propylene glycol placed in the impingers was based on the amount of test material (mg) being collected over a 6-hour exposure. The air control chamber was sampled weekly to validate the lack of test material in the control atmosphere.
After collecting a sample with the impingers, the collecting solution was quantitatively transferred to pre-weighted 20 mL scintillation vials and weighed again to determine the total material present. The solution was then sent for gas chromatographic analysis.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
2, 19, 129, 537 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
In addition, 2 males and 2 females were assigned to each group as health monitoring sentinels
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): computer randomization
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not reported
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Signs of intoxication, mortality, morbidity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes weekly


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No



OPHTHALMOSCOPIC EXAMINATION: No



HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (identity) Nembutal
- Animals fasted: Yes ; overnight
- How many animals: all surviving animals
- Parameters checked
hematocrit
hemoglobin
red blood cell
white blood cell count
platelet count
mean cell corpuscular volume
mean cell corpuscular hemoglobin
mean corpuscular hemoglobin conc.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked
triglyceride
cholesterol
blood urea nitrogen
glucose
alkaline phosphatase
total protein
albumin
chloride
sodium
potassium
globulin
albumin:globulin ratio
creatinine
bilirubin (total)
calcium
inorganic phosphorous
glutamic pyruvic transaminase/alanine aminotransferase activity
glutamic oxaloacetic transaminase/asparate aminotransferase activity
gamma-glutamyl transferase activity



URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the first day of exposure, and shortly after animals were removed from the chambers on the last day of exposure.
- Dose groups that were examined: all animals prior to exposure; all surviving animals on the last day of exposure
- Battery of functions tested: sensory activity / grip strength / motor activity / other: convulsions, tremors, stereotypic or bizzarre behavior


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Macroscopic: Gross examination included examination of the external surface of the body, all orifices and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY: Yes
- Microscopic: Tissues processed and examined microscopically will be collected, saved, weighed and fixed in 10% neutral buffered formalin. Tissues included but not limited to, the nasal cavity, liver, brain, kidney, spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, adrenals, spleen, trachea, larynx, heart, and gross lesions.
Other examinations:
Neurologic evaluations were performed the day prior to the first day of exposure, and shortly after animals were removed from the chambers on the last day of exposure.
Statistics:
One-way analysis of covariance models were used to assess the presence or absence of an overall compound effect. Separate one-way analyses were used within the male and female data to assess overall treatment group effects. Pairwise comparisons of least square means between control and each treatment level were evaluated using Dunnett's t-test. The criterion for statistical significance for all comparisons was p-value of 0.05 or less.

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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Time of death: all animals exposed to 537 mg/m3 died by exposure day 11; 1 during week 2 of dosing - Number of deaths at each dose: 10 at 537 mg/m3; 1 accidental death in control group; all animals exposed to 537 mg/m3 died by exposure day 11 and one control animal died during week 2 of exposure.
- Clinical signs: Animals exposed to 537 mg/m3 prior to death exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation, and lacrimation. At 129 mg/m3 transient occurrences of unstable gait, respiratory noise, salivation, and lacrimation were observed. Animals exposed at 19 and 2 mg/m3 showed no treatment-related signs of toxicity.


BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases in body weight occurred in 537 mg/m3 males during week 1, and in females during weeks 1 and 2. Males exposed to 129 mg/m3 showed statistically significant decreases in body weight and body weight change during weeks 2, 3, 4. The females exposed at 129 mg/m3 showed statistically significant decreases in body weight and body weight change during weeks 3 and 4. At 19 mg/m3 statistically significant decreases in body weight and body weight change were seen in the females during week 2. Females in the 2 mg/m3 exposure group showed statistically significant decreases in body weight and body weight change during weeks 2, 3, and 4.


FOOD CONSUMPTION
A statistically significant decrease in feed consumption was observed in all animals in the 537 mg/m3 exposure groups during week 1. A statistically significant decrease in the feed consumption for the 129 mg/m3 males was observed during weeks 2 and 3.


FOOD EFFICIENCY not examined


WATER CONSUMPTION not examined


OPHTHALMOSCOPIC EXAMINATION not examined


HAEMATOLOGY
No treatment-related effect on any hematologic parameter at any dose level. Animals exposed at 19 mg/m3 showed statistically significant decrease in mean cell hemoglobin and mean cell hemoglobin concentrations for the females. No changes in hematology parameters were seen at any other concentration. In the absence of a dose-response relationship, none of the above changes were judged treatment-related. No exposure-related differences in differential parameters were observed.

CLINICAL CHEMISTRY
- Clinical chemistry: No treatment-related clinical chemistry effects were observed in any group. Statistically significant decreases in total protein and globulin in males at 129 mg/m3. At 19 mg/m3 statistically significant decreases in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase in males and triglyceride in females. Males exposed at 2 mg/m3 showed a statistically significant decrease in creatinine and glutamic oxaloacetic transaminase; females in this group showed an increase in glutamic oxaloacetic transaminase. In the absence of a concentration-response relationship, none of the above changes were judged to be treatment-related.

URINALYSIS not examined


NEUROBEHAVIOUR


ORGAN WEIGHTS
No treatment-related organ weight changes observed at any dose level.


GROSS PATHOLOGY
Gross pathologic changes were observed only in animals exposed to 537 mg/m3. These changes consisted of partially or fully blocked nasal cavities and gas-filled stomachs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of male and female rats exposed to 537 mg/m3 showed changes in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithial ncerosis and inflammation of the submucosa. In addition, all meati and primarily the dorsal meati had exudates composed of mucous, fibrin, erythrocytes, exfoliated epithelium and inflammatory cells. The larynx and trachea had epithelial necrosis, submucosal inflammation an exudates in their lumens. Two female rats had foci of inflammation of bronchi, bronchioles and/or alveoli. Specific examination of tissues from the central (brain and spinal cord) and peripheral (sciatic nerve) nervous system as well as skeletal muscle from animals exposed to 537 mg/m3 revealed no changes indicative of neurotoxicity. The nasal cavity was the only target tissue in animals exposed to 129 mg/m3. The lesions were primary graded as slight and focal in nature. These lesions consisted of necrosis of respiratory and olfactory epithelium accompanied by inflammation in the mucosa and submucosa, as well as, exudates in meati which usually was overlying necrotic foci. Also observed were foci of respiratory epithelial hyperplasia and squamosus metaplasia (respiratory epithelium replaced by squamous epithelium). The olfactory epithelium and underlying Bowman's gland epithelium were atrophied. Rats exposed to 19 and 2 mg/m3 had not exposure-related microscopic changes.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) not applicable

OTHER: The sentinel animal serology results were negative for the presence of adventitious virus and bacteria antibodies.


Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
19 mg/m³ air
Sex:
male/female
Basis for effect level:
other: overall effects histopathology of the nasal cavities
Dose descriptor:
BMCL10
Effect level:
36.2 mg/m³ air
Sex:
male/female
Basis for effect level:
other: BMDL10 based on nasal cavity data, as this was the most sensitive effect (Multistage model)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Inhalation exposure of the test material produced 100% mortality at 537 mg/m3. Prior to the death, these animals exhibited labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation, lacrimation, and decresed body weight, body weight gain and feed consumption. At 129 mg/m3, transient occurrences of unstable gait, respiratory noise, salivation, lacrimation, and slight decreased body weight gains and feed consumption were observed.

No treatment-related signs were observed at 19 and 2 mg/m3. Organ weights and blood analysis revealed no treatment-related effects. At the end of the four-week exposure period, neurological evalutions of all surviving animals showed no signs of a cumulative toxic effect on the nervous system in any group. On the basis of the most sensitive indictor of toxicity, the histopathological changes seen in the nasal cavities, the NOEL for a four-week nose-only inhalation exposure of the test material was 19 mg/m3.

The response of 0 0 0 2 10 was the most sensitive therefore the BMD value of 80.96 mg/m3 and the BMDL value of 36.20 mg/m3 are chosen corresponding to the multistage model with the lowest AIC.
Executive summary:

Five groups, designated 1,2,3,4 and 5, each containing 10 male and 10 female Crl:CD BR rats, received nose-only inhalation exposures for 6 hours per day, 5 days a week, for four weeks to air or vapors of the test material. Group 1 animlas were exposed to filtered air only. Groups 2,3,4 and 5 were exposed to 2, 19, 129, and 537 mg/m3 of the test material, respectively. All surviving animals from each group were necropsied at the end of the four week exposure period. Body weight, feed consumption, clinical signs, neurology, clinical chemistry, hematology, organ weight and histopathology evaluations were performed on all surviving animals during this study.

All animlas exposed to 537 mg/m3 died by exposure day 11. Prior ro death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation. At 129 mg/m3 transient occurrences of unstable gait, respiratory noise, salivation and lacrimation were observed. Animals exposed to 19 mg/m3 showed no treatment related signs of toxicity, with the exception of one incident of lacrimation. No treatment-related signs were observed at 2 mg/m3. At the end of the four-week exposure period, neurological evaluations of all surviving animals showed no effect on the nervous system in any group.

Treatment -related decreases in body weight and feed consumption was observed in both sexes at 537 mg/m3. Animlas exposed to 129 mg/m3 showed a decrease in body weights for both sexes, and a decrease in feed consumption in males. Animlas exposed to 19 and 2 mg/m3 showed no treatment-related changes in body weight or feed consumption.

No treatment-related effects were seen in any group for clinical chemistry, hematology or organ weights.

Gross pathologic changes were observed only in animals exposed to 537 mg/m3. These changes consisted of partially or fully blocked nasal cavities and gas-filled stomachs. Microscopic examination of male and female rats exposed to 537 mg/m3 showed chnages in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithelial necrosis and inflammation. The larynx and trachea had epithelial necrosis and inflammation. The lungs of two females showed foci of inflammation in the bronchi, bronchioloes and/or alveoli. Male and female rats exposed to 129 mg/m3 showed slight focal lesions of the nasal cavity. Rats exposed to 19 or 2 mg/m3 had no exposure related nasal cavity microscopic changes.

On the basis of the most sensitive indicator of toxicity, the histopathological changes seen in the nasal cavities, the no observed effect level (NOEL) following four weeks of nose-only inhalation exposure to the test material was 19 mg/m3.

A benchmark dose (BMD) analysis of relevant toxicity data for primene was conducted in order to establish BMD and BMDL (lower limit of the 95% confidence interval on the BMD) values. The U.S. EPA’s Benchmark Dose Software (BMDS) was used for the analysis (U.S. EPA 2009). Data from a four week vapor inhalation study in rats was used (Hagan et al., 1994). The exposure doses were 0, 2, 19, 129, and 537 mg/cubic meter of primene.

The key endpoints evaluated were body weight at week four (dose 537 not used due to deaths before the end of the study) and the nasal cavity with both the respiratory and olfactory epithelium. The study report indicated the nasal cavity was the most sensitive target organ with histopathologic changes, particularly desquamation, necrosis and inflammation.

The results for models for a particular response were all within a factor of 3 so the model with the lowest AIC is the most desirable. The plots show that the fits for the models for incidence 0 0 0 2 10 were all very similar except the quantal linear model which had a poorer fit than the others. The response of 0 0 0 2 10 was the most sensitive therefore the BMD value of 80.96 mg/m3 and the BMDL value of 36.20 mg/m3 are chosen corresponding to the multistage model with the lowest AIC.