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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Linalool
- Physical state: clear, colorless liquid
- Analytical purity: 99.6%
- Batch No.: 1SB101
- Expiration date of the batch: 19 October 2013
- Stability under test conditions: stability was confirmed during the experiment
- Storage condition of test material: in a sealed container at room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 196 - 243 g (males) and 156 - 193 g (females)
- Fasting period: during acclimation to the nose-only restraint and the exposure periods (no food and water), during the period prior to necropsy (no food)
- Housing: individually in clean, stainless steel, wire-mesh cages
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal); ad libitum
- Water: reverse osmosis-treated (on-site) drinking water; ad libitum
- Acclimation period: minimum of 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes: minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: inhalation: combined aerosol and vapor
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: During method development, particle size measurements were attempted using brass impactors with glass fiber filters for substrates and using a stainless steel impactor with stainless steel substrates. Based on the results of deposition on these substrates, there is insufficient aerosol to accurately quantify aerosol particle size. This is presumed to be because of the physical properties of the test substance at the target exposure levels of 0.63 mg/m3 and 63 mg/m3.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: (7.9-L) Stainless-steel conventional nose-only exposure systems with synthetic rubber grommets in exposure ports
- System of generating particulates/aerosols: Test substance was supplied to a single-jet Collison nebulizer using a Harvard syringe pump and a 50-mL syringe. The resulting test substance atmosphere from the nebulizer (combined aerosol and vapor) was delivered to a 4.8 L chromatography jar and mixed with dilution air prior to delivery to each exposure system.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations of the test material (combined vapor and aerosol) within each nose-only system were determined using an appropriate gas chromatography (GC) method. Concentrations were determined at approximately 60 minute intervals for each test material exposure system and at least once daily for the control system.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 days per week (10 total exposures for each animal)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.63, 6.3 and 63 mg/m3 (equivalent to 0.1, 1 and 10 ppm)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.76, 6.6, 56 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
MORTALITY AND MORIBUNDITY: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
on days of exposure: prior to exposure, at the approximate midpoint of each exposure, and 1 hour post-exposure
on non-exposure days: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first exposure, twice weekly during exposure and day before the scheduled necropsy

FOOD CONSUMPTION:
- Time schedule: twice weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes


HAEMATOLOGY - COAGULATION PARAMETERS: Yes
- Time schedule for collection of blood: at time of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight fasting period)
- How many animals: all animals
Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobinconcentration, Platelet count, Prothrombin time , Activated partial thromboplastin time, Reticulocyte count, Mean platelet volume, Differential leukocyte count, Red cell distribution width, Hemoglobin distribution width, Platelet estimate, Red cell morphology


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at time of necropsy
- Animals fasted: Yes (overnight fasting period)
- How many animals: all animals
Albumin, Total protein, Globulin, Albumin/globulin ratio, Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase , Aspartate aminotransferase , Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Sorbitol dehydrogenase, Lactate dehydrogenase , Hemolysis, Lipemia, Icterus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

BRONCHOALVEOLAR LAVAGE (BAL): Yes
- Time schedule: at necropsy
- How many animals: 5 animals/sex/group (BALF animals)
- Examined parameters: lactate dehydrogenase, total protein, alkaline phosphatase, Total and differential cell counts (Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils), cytokines (TNF-α, IL-5, IL-10, ICAM-1, IFN-γ, IL-4, TGF-β, MCP-1, IL-1β, IL-13, MIP-2, RANTES)


SERUM CYTOKINE EVALUATION:
- Time schedule: at necropsy
- How many animals: 5 animals/sex/group (BALF animals)
- samples were taken, but not further assessed for cytokines


Sacrifice and pathology:
On the day following the final exposure, all animals were sacrificed and subjected to necropsy. Animals were subjected to either tissue
collection (5 animals/sex/group; histopathology animals) or bronchoalveolar lavage (5 animals/sex/group; BALF animals).

GROSS PATHOLOGY and HISTOPATHOLOGY: Yes
- selected organs were weighed and selected tissues were examined microscopically from 5 animals/sex/group (histopathology animals) at the scheduled necropsy
The following organs were weighed from all animals at the scheduled necropsy: Adrenals, Brain, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids

Microscopic examination of the lungs (with bronchi), BALT, nasal tissues, NALT, larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions were performed for 5 animals/sex/group not assigned for the BAL at the scheduled necropsy. In addition, microscopic examination was performed on the kidneys and liver from 5 animals/sex in the control and 63 mg/m3 (high exposure level) groups at the scheduled necropsy. For the nasal cavity and turbinates, 6 sections were examined, including a section containing the nasopharyngeal duct. Three levels of the larynx, including 1 at the base of the epiglottis, were examined. At least 2 sections of the trachea were examined, including 1 longitudinal section through the carina of the extrapulmonary bifurcation of bronchi and 1 transverse section taken at approximately the midpoint of the trachea.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, clinical pathology, and organ weight data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled necropsy. There were no test substance-related clinical observations.

BODY WEIGHT AND WEIGHT GAIN
Body weights were unaffected by test substance exposure.

FOOD CONSUMPTION
Food consumption was unaffected by test substance exposure.

HAEMATOLOGY AND COAGULATION
There were no alterations in hematology and coagulation parameters that were associated
with test substance exposure

CLINICAL CHEMISTRY
There were no alterations in serum chemistry parameters that were associated with test
substance exposure.


ORGAN WEIGHTS
There were no test substance-related alterations in organ weights.

GROSS PATHOLOGY
There were no test substance-related macroscopic findings at the scheduled necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Nonadverse, test substance-related microscopic findings were noted in the nasal cavity of males and females exposed to 0.63, 6.3, and 63 mg/m3 of the test substance:

Subacute inflammation and/or squamous epithelial hyperplasia were observed in control and test substance-exposed males and females (see attachment). There was a higher incidence of subacute inflammation in test substance-exposed males and females and a higher incidence of squamous epithelial hyperplasia in test substance-exposed males. There were nasal level 1 sections that contained areas of transitional epithelium along the dorsal meatus/septum and turbinates. Hyperplasia of transitional epithelium were observed in nasal level 1 of control and test substance-exposed males and females with a similar incidence in control and test substance-exposed groups but with a slightly higher severity in test substance-exposed males and females. Transitional and squamous epithelial degeneration had variable incidences and severities in control and test substance-exposed
males and females.
Transitional epithelial hyperplasia and/or degeneration and subacute inflammation in nasal level 2 and subacute inflammation in nasal level 3 were observed in control and test substance-exposed males and females (all exposure groups). There was a similar incidence and severity between control and test substance-exposed groups in nasal levels 2 and 3 of males and nasal level 2 of females. There was a slightly higher incidence of subacute inflammation in nasal level 3 of test substance-exposed females (all exposure groups).
Evaluation:
Test substance-related inflammation and epithelial (squamous and transitional) hyperplasia in nasal level 1 of males and females and subacute inflammation of nasal level 3 in females were considered exacerbated background lesions as they were also observed in control group males and females, and were not considered adverse. Other epithelial findings in nasal level 1 of males and females, inflammation, and/or epithelial changes in nasal levels 2 and 3 in males and nasal level 2 in females had similar incidences in control and test substance exposed groups.

OTHER FINDINGS
CYTOKINE AND BRONCHOALVEOLAR LAVAGE FLUID CLINICAL PATHOLOGY EVALUATIONS
There were no alterations in BALF chemistry parameters and cytology that were associated with test substance exposure.
There were no alterations in BALF cytokine levels that were associated with test substance exposure.

Effect levels

Dose descriptor:
NOAEC
Effect level:
63 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: nonadverse microscopic findings in the nasal cavity observed

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion